Transformation system in the field of filamentous fungal hosts

ABSTRACT

A novel transformation system in the field of filamentous fungal hosts for expressing and secreting heterologous proteins or polypeptides is described. The invention also covers a process for producing large amounts of polypeptide or protein in an economical manner. The system comprises a transformed or transfected fungal strain of the genus  Chrysosporium,  more particularly of  Chrysosporium lucknowense  and mutants or derivatives thereof. It also covers transformants containing  Chrysosporium  coding sequences, as well expression-regulating sequences of  Chrysosporium  genes. Also provided are novel fungal enzymes and their encoding sequences and expression-regulating sequences.

REFERENCE TO PRIOR APPLICATIONS

[0001] This is a continuation-in-part of international applicationPCT/NL99/006 18, filed Oct. 6, 1999, which is a continuation-in-part ofinternational application PCT/EP98/06496, filed Oct. 6, 1998.

SUMMARY OF THE INVENTION

[0002] The subject invention pertains to a novel transformation systemin the field of filamentous fungal hosts for expressing and secretingheterologous proteins or polypeptides. The invention also covers aprocess for producing large amounts of polypeptide in an economicalmanner. The system comprises a transformed or transfected fungal strainof the genus Chrysosporium, more particularly of Chrysosporiumlucknowense and mutants or derivatives thereof. It also coverstransformants containing Chrysosporium coding sequences. Novel mutantChrysosporium strains are disclosed as are novel enzymes derivedtherefrom. The subject invention further relates to novel enzymesderived from filamentous fungi, especially from strains of the genusChrysosporium, and to coding sequences and expression-regulatingsequences for these enzymes.

BACKGROUND TO THE INVENTION

[0003] A number of hosts for gene expression and methods oftransformation have been disclosed in the prior art. Bacteria are oftenmentioned e.g. Escherichia coli. E. coli is however a micro-organismincapable of secretion of a number of proteins or polypeptides and assuch is undesirable as host cell for production of protein orpolypeptide at the industrial level. An additional disadvantage for E.coli, which is valid also for bacteria in general, is that prokaryotescannot provide additional modifications required for numerous eukaryoticproteins or polypeptides to be produced in an active form. Glycosylationof proteins and proper folding of proteins are examples of processingrequired to ensure an active protein or polypeptide is produced. Toensure such processing one can sometimes use mammalian cells; however,the disadvantage of such cells is that they are often difficult tomaintain and require expensive media. Such transformation systems aretherefore not practical for production of proteins or polypeptides atthe industrial level. They may be cost efficient for highly pricedpharmaceutical compounds requiring relatively low amounts, but certainlynot for industrial enzymes.

[0004] A number of fungal expression systems have been developed e.g.Aspergillus niger, Aspergillus awamori, Aspergillus nidulans,Trichoderma reesei. A number of others have been suggested but forvarious reasons have not found wide-spread acceptance or use. In generalterms the ideal host must fulfil a large number of criteria:

[0005] The ideal host must be readily fermented using inexpensivemedium.

[0006] The ideal host should use the medium efficiently.

[0007] The ideal host must produce the polypeptide or protein in highyield, i.e. must exhibit high protein

[0008] The ideal host should be capable of efficient secretion of theprotein or polypeptide.

[0009] The ideal host must enable ease of isolation and purification ofthe desired protein or polypeptide.

[0010] The ideal host must process the desired protein or polypeptidesuch that it is produced in an active form not requiring additionalactivation or modification steps.

[0011] The ideal host should be readily transformed.

[0012] The ideal host should allow a wide range of expression regulatoryelements to be used thus ensuring ease of application and versatility.

[0013] The ideal host should allow use of easily selectable markers thatare cheap to use.

[0014] The ideal host should produce stable transformants.

[0015] The ideal host should allow cultivation under conditions notdetrimental to the expressed protein or polypeptide e.g. low viscosity,low shear.

[0016] Fungal systems that have not yet found widespread use aredescribed e.g. in U.S. Pat. No. 5,578,463 by Berka et al suggestingNeurospora, Podospora, Endothia, Mucor, Cochoibolus and Pyriculariatogether with Aspergillus and Trichoderma. However only illustrations oftransformation and expression are provided for Aspergillus andTrichoderma and no details are provided for any of the other suggestedhosts.

[0017] 1 WO 96/02563 and U.S. Pat. Nos. 5,602,004, 5,604,129 and5,695,985 to Novo Nordisk describe the drawbacks of Aspergillus andTrichoderma systems and suggests cultivation conditions for other fungimay be more suited to large scale protein production. The only examplesprovided for any transformed cultures are those of Myceliophthorathermophila, Acremonium alabamense, Thielavia terrestris andSporotrichum cellulophilum strains. The Sporotrichum strain is reportedto lyse and produce green pigment under fermentation conditions notleading to such results for the other strains. A non-sporulating mutantof Thielavia terrestris is described as being the organism of choice byvirtue of its morphology. However it is also stated that theprotoplasting efficiency of Thielavia and Acremonium (whereby theAcremonium strain used was the imperfect state of the Thielavia strainused) is low and that hygromycin was not useful as a selection marker. Alarge number of others are suggested as being potentially useful byvirtue of their morphology but no transformation thereof is described.The suggested strains are Corynascus, Thermoascus, Chaetomium,Ctenomyces, Scytalidium and Talaromyces. The transformed hosts arementioned as only producing low levels of the introduced Humicolaxylanase with Thielavia producing the lowest amount; however, theinformation is ambiguous and could actually infer Thielavia was the bestembodiment. The nomenclature of this reference is based on the ATCCnames of Industrial Fungi of 1994. Thus it is apparent no high degree ofheterologous expression was achieved and in fact no positive correlationcould be derived between the postulated morphology and the degree ofexpression. If any correlation could be made, it was more likely to benegative. According to the 1996 ATCC fungal classification Sporotrichumthermophilum ATCC 20493 is a Myceliophthora thermophila strain.Currently the strain is still identified as Myceliophthora thermophila.The unpredicatability of the art is apparent from these recentdisclosures.

[0018] Also Allison et al (Curr. Genetics 21:225-229,1992) describedtransformation of Humicola grisea var. thermoidea using the lithiumacetate method and a Humicola enzyme-encoding sequence, but no report ofexpression of heterologous protein from such a strain has been provided.

[0019] In 1997 a patent issued to Hawaii Biotechnology Group fortransformed Neurospora for expression of mammalian peptide such aschymosin. The transformation of auxotrophic Neurospora crassa occurredwith spheroplasts. Endogenous transcriptional regulatory regions wereintroduced and cotransformation was carried out. Nothing is mentionedconcerning other hosts and other transformation protocols. Nothing isapparent from the disclosure concerning the degree of expression. It isdoubtful whether the degree of expression is high, as immunotechniques(which are useful for detecting small amounts of protein) are the onlytechniques used to illustrate the presence of the protein. No actualisolation of the protein is disclosed.

[0020] WO 97/26330 of Novo Nordisk suggests a method of obtainingmutants of filamentous fungal parent cells having an improved propertyfor production of heterologous polypeptide. The method comprises firstfinding a specific altered morphology followed by assessing whether atransformant produces more heterologous polypeptide than the parent. Themethod is illustrated only for strains of Fusarium A3/5 and Aspergillusoryzae. The method is suggested to be applicable for Aspergillus,Trichoderma, Thielavia, Fusarium, Neurospora, Acremonium, Tolyplocadium,Humicola, Scytalidium, Myceliophthora or Mucor. As stated above theunpredictability in the art and also the unpredictability of the methodof the cited application do not provide a generally applicable teachingwith a reasonable expectation of success.

DETAILED DESCRIPTION OF THE INVENTION

[0021] We now describe an alternative fungal expression system with thesimplicity of use of the above-mentioned Aspergilli and Trichodermafulfilling the above requirements. The new system has not been taught orsuggested in the prior art. The new system according to the inventionprovides the additional advantages that transformation rates are higherthan those for the frequently used Trichoderma reesei system. Inaddition the culture conditions offer the additional bonus of beingadvantageous for the expressed polypeptide.

[0022] We further describe a number of industrially interesting enzymesderived from the novel expressing system, together with full sequenceinformation. We also describe novel promoter systems derived fromChrysosporium strains and useful for expressing homologous andheterologous genes.

[0023] The present invention is thus also concerned with glycosylhydrolases of the families 7 (e.g. cellobiohydrolases), 10 (e.g.xylanases) and 12 (e.g. endoglucanases), and glyceraldehyde phosphatedehydrogenases, as identified by their amino acid sequence, as well aspeptides derived from these enzymatic proteins, and with nucleic acidsequences encoding these peptides and proteins, as well as, inparticular, with regulating sequences related to these genes.

[0024] In particular, the present invention pertains to isolated orrecombinant enzymic proteins or active parts thereof of the four classesreferred to above, including mutants thereof having at least a certaindegree of sequence identity as specified in the further disclosure andin the claims, as well as nucleic acid sequences encoding these proteinsor parts thereof, and/or nucelic acid sequences regulating theirexpression. These enzymes are especially: (1) a glycosyl hydrolase offamily 7 (cellobiohydrolase, CBH1) having at least 75%, preferably atleast 80% or even at least 85% amino acid identity with the sequence ofSEQ ID No 1; (2) a glycosyl hydrolase of family 10 (endoxylanase XYLF orXYL1) having at least 70%, preferably at least 75% or even at least 80%amino acid identity with the sequence of SEQ ID No 2; (3) a glycosylhydrolase family of 12 (endoglucanase, EG3) having at least 65%,preferably at least 70% or even at least 80% amino acid identity withthe sequence of SEQ ID No. 3; and (4) a glyceraldehyde phosphatedehydrogenase (GPD1) having at least 86%, preferably at least 90% oreven at least 93% amino acid identity with the sequence of SEQ ID No 4.Polypeptides and nucleic acid sequences encoding these polypeptides,having at least 20, preferably at least 30 contiguous amino acids of SEQID No's 1-4 are also a preferred part of the invention.

[0025] The recombinant enzymes may comprise essentially the completeprotein, or a truncated protein having at least part of the enzymaticactivity. Such truncated part may be the catalytic domain, or at leastabout 75% of the amino acids thereof. By way of example, the catalyticdomain of the CBH1 according to the invention comprises the aminoacids20-495 of the aminoacid sequence of SEQ ID No. 1, and the catalyticdomain of the XYL1 according to the invention comprises the aminoacids54-384 of the aminoacid sequence of SEQ ID No. 2. The catalytic domainmay or may not be combined with a signal sequence originating fromanother protein and/or with a carbohydrate-binding domain from anotherenzymic protein. Alternatively, the cellulose-binding domain of theenzymes of the invention (CBH1 and XYL1) may be fused to catalyticdomains of other enzymic proteins.

[0026] The nucleic acid sequences according to of the invention may becomplete protein-encoding regions or oligonucleotides or,preferentially, expression-regulating sequences. Oligonucleotides may beused also as probes for identifying genes corresponding to, but notidentical to the genes of SEQ ID No.'s 1-4; these genes, when fulfillingthe percentage identity criteria defined herein, as well as encoding andnon-encoding parts thereof and their expression products are also partof the invention.

[0027] The invention also pertains to expression systems (cassettes)comprising either an expression-regulatingregion (including a promoter)of any of the four protein classes fused to a gene encoding anotherprotein of interest, or an encoding region of any of these proteinsfused to another expression regulating region, or both theexpression-regulatingregion and the protein-encodingregion of thesenovel proteins. The expression-regulatingregion comprises at least 60%,preferably at least 70%, more preferably at least 75% or even 80% of the5′-non-coding region of SEQ ID No.'s 1-4, and/or at least 20, especiallyat least 40 contiguous nucleotides from these 5′ non-coding regions.Terminating sequences similarly derived from the 3″ non-coding regionsof the genes of the invention are also useful in expressing cassettes,whether combined with homologous or heterologous genes.

[0028] These expression systems may be contained in a Chrysosporiumhost, such as a Chrysosporium lucknowense host, or in another non-fungalor, preferably, fungal host. Examples of other fungal hosts are otherChrysosporium species or strains, Fusarium species, Aspergillus speciesetc. Such host may be advantageously a host that does not itself,intrinsically or as a result of the culture conditions, produce aprotein corresponding to the protein of interest, so as to simplify therecovery of the protein of interest.

[0029] Where reference is made in this specification and in theappending claims to “polypeptides” or “peptides” or “polypeptides ofinterest” or “peptides of interest” as the products of the expressionsystem of the invention, this term also comprise proteins, i.e.polypeptides having a particular function and/or secondary and/ortertiary structure. Where reference is made to percentage amino acididentity, such identity relates to e complete protein or a to a specificpart defined by initial and final amino acid number, as determined bythe conventionally used BLAST algorithm.

[0030] In the production method of the invention, the pH of the culturemedium can be neutral or alkaline thus no longer subjecting the producedprotein or polypeptide to aggressive and potentially inactivating acidpH. It is also possible to culture at acid pH such as pH 4 for caseswhere the protein or polypeptide is better suited to an acidicenvironment. Suitably culture can occur at a pH between 4.0-10.0. Apreference however exists for neutral to alkaline pH as the host strainexhibits better growth at such pH, e.g. between 6 and 9. Growth atalkaline pH which can be from pH 8 up and can even be as high as 10 isalso a good alternative for some cases. Also the cultivation temperatureof such host strains is advantageous to the stability of some types ofproduced polypeptide. The cultivation temperature is suitably at atemperature of 25-43° C. A temperature in the range from 40° C. down to23° C. or 30° C. is also advantageously applied. Clearly such conditionsare of particular interest for production of mammalian polypeptides. Theselected temperature will depend on cost effectiveness of thecultivation and sensitivity of the polypeptide or cultivation strain.The conditions will be determined by the skilled person without undueburden on a case-by-case basis, as is common in the art.

[0031] It has also been ascertained that the biomass to viscosityrelation and the amount of protein produced is exceedingly favourablefor the host according to the invention. Comparisons have been carriedout with Trichoderma longibrachiatum (formerly also known asTrichodermareesei) and with Aspergillus niger. Trichodermalongibrachiatum gave 2.5-5 g/l biomass, Aspergillus niger gave 5-10 g/lbiomass and the host according to the invention gave 0.5-1 g/l biomassunder their respective optimised conditions. This thus offers 5-10 foldimprovement over the commercially used strains. These commercial strainsare strains which themselves are considered in the art to be highproducers of proteins and they are successfully used for commercialprotein production. They have been cultured under their optimalconditions developed and run viably in large-scale commercialfermenters. The same strains were used to illustrate enormousimprovement in viscosity values for cultures of the host according tothe invention. At the end of the fermentation process Trichodermalongibrachiatum gave a value of 200-600 cP (Centipoise), Aspergillusniger gave a value of 1500-2000 cP and the host according to theinvention gave a value below 10 cP. This thus provides at least 20-200fold improvement for viscosity values over the commercially usedstrains. A quite surprising further aspect was that the protein levelsdetermined for the host cells according to the invention were muchhigher than for the commercial Aspergilli and Trichoderma reeseistrains, even with the above mentioned surprisingly low biomass andviscosity levels. In summary an easy to use versatile improvedtransformation system and expression system with improved culturingconditions has hereby been introduced. The strains according to theinvention produce surprisingly higher protein levels under theseimproved conditions and in addition they do such in a shorterfermentertime.

[0032] The subject invention is directed at mutant Chrysosporium strainscomprising a nucleic acid sequence encoding a heterologous protein orpolypeptide, said nucleic acid sequence being operably linked to anexpression regulating region and optionally a secretion signal encodingsequence and/or a carrier protein encoding sequence. Preferably arecombinant strain according to the invention will secrete thepolypeptide of interest. This will avoid the necessity of disrupting thecell in order to isolate the polypeptide of interest and also minimisethe risk of degradation of the expressed product by other components ofthe host cell.

[0033]Chrysosporium can be defined by morphology consistent with thatdisclosed in Barnett and Hunter 1972, Illustrated Genera of ImperfectFungi, 3rd Edition of Burgess Publishing Company. Other sourcesproviding details concerning classification of fungi of the genusChrysosporium are known e.g. Sutton Classification (Van Oorschot, C. A.N. (1980) “A revision of Chrysosporium and allied genera” in Studies inMycology No. 20 of the CBS in Baarn The Netherlands p1-36). CBS is oneof the depository institutes of the Budapest Treaty. According to theseteachings the genus Chrysosporium falls within the family Moniliaceaewhich belongs to the order Hyphomycetales. The criteria that can be usedare the following:

[0034] 1. Signs of Hyphomycetales Order:

[0035] Conidia are produced directly on mycelium, on separatesporogenous cells or on distinct conidiophores.

[0036] 2. Signs of Moniliaceae Family:

[0037] Both conidia and conidiophores (if present) are hyaline orbrightly coloured; conidiophores are single or in loose clusters.

[0038] 3. Signs of Chrysosporium Corda 1833 Genus:

[0039] Colonies are usually spreading, white, sometimes cream-coloured,pale brown or yellow, felty and/or powdery. Hyphae are mostly hyalineand smooth-walled, with irregular, more or less orthotopic branching.Fertile hyphae exhibit little or no differentiation. Conidia areterminal and lateral, thallic, borne all over the hyphae, sessile or onshort protrusions or side branches, subhyaline or pale yellow, thin- orthick-walled, subglobose, clavate, pyriform, orobovoid, 1-celled, rarely2-celled, truncate. Intercalary conidia are sometimes present, aresolitary, occasionally catenate, subhyaline or pale yellow, broader thanthe supporting hyphae, normally 1-celled, truncate at both ends.Chlamydosporesare occasionallypresent.

[0040] Another source providing information on fungal nomenclature isATCC (US). Their website is http://www.atcc.org. CBS also has a website(http//www.cbs.knaw.nl) providing relevant information. VKM in Moscow isalso a reliable source of such information; the e-mail address for VKMis http://www.bdt.org.br.bdt.msdn.vkm/general. Another source ishttp//NT.ars-grin.gov/-fungaldatabases. All these institutions canprovide teaching on the distinguishing characteristics of aChrysosporium.

[0041] Strains defined as being of Myceliophthora thermophila are notconsidered to define Chrysosporium strains according to the definitionof the invention. In the past there has been considerable confusion overthe nomenclature of some Myceliophihora strains. Preferably theChrysosporium according to the invention are those which are clearlydistinguishable as such and cannot be confused with Myceliophthora,Sporotrichum or Phanerochaete chrysosporium.

[0042] The following strains are defined as Chrysosporium but thedefinition of Chrysosporium is not limited to these strains: C.botryoides, C. carmichaelii, C. crassitunicatum, C. europae, C.evolceannui, C. farinicola, C. fastidium, C. filiforme, C. georgiae, C.globiferum, C. globiferum var. articulatum, C. globiferum var. niveum,C. hirundo, C. hispanicum, C. holmii, C. indicum, C. inops, C.keratinophilum, C. kreiselii, C. kuzurovianum, C. lignorum, C. lobatum,C. lucknowense, C. lucknowense Garg 27K, C. medium, C. medium var.spissescens, C. mephiticum, C. merdarium, C. merdarium var. roseum, C.minor, C. pannicola, C. parvum, C. parvum var. crescens, C. pilosum, C.pseudomerdarium, C. pyriformis, C. queenslandicum, C. sigleri, C.sulfureum, C. synchronum, C. tropicum, C. undulatum, C. vallenarense, C.vespertilium, C. zonatum.

[0043]C. lucknowense forms one of the species of Chrysosporium that haveraised particular interest as it has provided a natural high producer ofcellulase proteins (WO 98/15633 and related U.S. Pat. No. 5,811,381, aswell as U.S. Pat. No. 6,015,707). The characteristics of thisChrysosporium lucknowense are:

[0044] Colonies attain 55 mm diameter on Sabouraud glucose agar in 14days, are cream-coloured, felty and fluffy; dense and 3-5 mm high;margins are defined, regular, and fimbriate; reverse pale yellow tocream-coloured. Hyphae are hyaline, smooth- and thin-walled, littlebranched. Aerial hyphae are mostly fertile and closely septate, about1-3.5 μm wide. Submerged hyphae are infertile, about 1-4.5 μm wide, withthe thinner hyphae often being contorted. Conidia are terminal andlateral, mostly sessile or on short, frequently conical protrusions orshort side branches. Conidia are solitary but in close proximity to oneanother, 1-4 conidia developing on one hyphal cell, subhyaline, fairlythin- and smooth-walled, mostly subglobose, also clavate orobovoid,1-celled, 2.5-11×1.5-6 μm, with broad basal scars (1-2 μm). Intercalaryconidia are absent. Chlamydospores are absent. ATCC 44006, CBS 251.72,CBS 143.77 and CBS 272.77 are examples of Chrysosporium lucknowensestrains and other examples are provided in WO 98/15633 and U.S. Pat. No.5,811,381.

[0045] A further strain was isolated from this species with an evenhigher production capacity for cellulases. This strain is called C1 byits internal notation and was deposited with the InternationalDepository of the All Russian Collection of micro-organisms of theRussian Academy of Sciences Bakrushina Street 8, Moscow, Russia 113184on Aug. 29, 1996, as a deposit according to the Budapest Treaty and wasassigned Accession Number VKM F-3500D. It is called Chrysosporiumlucknowense Garg 27K. The characteristics of the C1 strain are asfollows:

[0046] Colonies grow to about 55-66 mm diameter in size onpotato-dextrose agar in about 7 days; are white-cream-coloured, felty,2-3 μm high at the center; margins are defined, regular, fimbriate;reverse pale, cream-coloured. Hyphae are hyaline, smooth- andthin-walled, little branched. Aerial hyphae are fertile, septate, 2-3 mmwide. Submerged hyphae are infertile. Conidia are terminal and lateral;sessile or on short side branches; absent; solitary, but in closeproximity to one another, hyaline, thin- and smooth-walled, subglobose,clavate or obovoid, 1-celled, 4-10 μm. Chlamydo-spores are absent.Intercalaryconidia are absent.

[0047] The method of isolation of the C1 strain is described in WO98/15633 and U.S. Pat. No. 5,811,381. Strains exhibiting such morphologyare included within the definition of Chrysosporium according to theinvention. Also included within the definition of Chrysosporium arestrains derived from Chrysosporium predecessors including those thathave mutated somewhat either naturally or by induced mutagenesis. Inparticular the invention covers mutants of Chrysosporium obtained byinduced mutagenis, especially by a combination of irradiation andchemical mutagenesis.

[0048] For example strain C1 was mutagenised by subjecting it toultraviolet light to generate strain UV13-6. This strain wassubsequently further mutated with N-methyl-N′-nitro-N-nitrosoguanidinetogenerate strain NG7C-19. The latter strain in turn was subjected tomutation by ultraviolet light resulting in strain UVi8-25. During thismutation process the morphological characteristics have varied somewhatin culture in liquid or on plates as well as under the microscope. Witheach successive mutagenesis the cultures showed less of the fluffy andfelty appearance on plates that are described as being characteristic ofChrysosporium, until the colonies attained a flat and matted appearance.A brown pigment observed with the wild type strain in some media wasalso less prevalent in mutant strains. In liquid culture the mutantUV18-25 was noticeably less viscous than the wild type strain C1 and themutants UV13-6 and NG7C-19. While all strains maintained the grossmicroscopic characteristics of Chrysosporium, the mycelia becamenarrower with each successive mutation and with UV18-25 distinctfragmentation of the mycelia could be observed. This mycelialfragmentation is likely to be the cause of the lower viscosityassociated with cultures of UV18-25. The ability of the strains tosporulate decreased with each mutagenic step. The above illustrates thatfor a strain to belong to the genus Chrysosporium there is some leewayfrom the above morphological definition. At each mutation stepproduction of cellulase and extracellular proteins has in addition alsoincreased, while several mutations resulted in decrease of proteaseexpression. Criteria with which fungal taxonomy can be determined areavailable from CBS, VKMF and ATCC for example.

[0049] In particular the anamorph form of Chrysosporium has been foundto be suited for the production application according to the invention.The metabolism of the anamorph renders it extremely suitable for a highdegree of expression. A teleomorph should also be suitably as thegenetic make-up of the anamorphs and teleomorphs is identical. Thedifference between anamorph and teleomorph is that one is the asexualstate and the other is the sexual state. The two states exhibitdifferent morphology under certain conditions.

[0050] It is preferable to use non-toxic Chrysosporium strains of whicha number are known in the art as this will reduce risks to theenvironment upon large scale production and simplify productionprocedures with the concomitant reduction in costs.

[0051] An expression-regulatingregion is a DNA sequence recognised bythe host Chrysosporium strain for expression. It comprises a promotersequence operably linked to a nucleic acid sequence encoding thepolypeptide to be expressed. The promoter is linked such that thepositioning vis-á-vis the initiation codon of the sequence to beexpressed allows expression. The promoter sequence can be constitutiveor inducible. Any expression regulating sequence or combination thereofcapable of permitting expression of a polypeptide from a Chrysosporiumstrain is envisaged. The expression regulating sequence is suitably afungal expression-regulating region e.g. an ascomycete regulatingregion. Suitably the fungal expression regulating region is a regulatingregion from any of the following genera of fungi: Aspergillus,Trichoderma, Chrysosporium (preferred), Hansenula, Mucor, Pichia,Neurospora, Tolypocladium, Rhizomucor, Fusarium, Penicillium,Saccharomyces, Talaromyces or alternative sexual forms thereof likeEmericella, Hypocrea e.g. the cellobiohydrolase promoter fromTrichoderma, glucoamylase promoter from Aspergillus, glyceraldehydephosphate dehydrogenase promoter from Aspergillus, alcohol dehydrogenaseA and alcohol dehydrogenase R promoter of Aspergillus, TAKA amylasepromoter from Aspergillus, phosphoglycerate and cross-pathway controlpromoters of Neurospora, aspartic proteinase promoter of Rhizomucormiehei, lipase promoter of Rhizomucor miehei and beta-galactosidasepromoter of Penicillium canescens. An expression regulating sequencefrom the same genus as the host strain is extremely suitable, as it ismost likely to be specifically adapted to the specific host. Thuspreferably the expression regulating sequence is one from aChrysosporium strain.

[0052] We have found particular strains of Chrysosporium to expressproteins in extremely large amounts and natural expression regulatingsequences from these strains are of particular interest. These strainsare internally designated as Chrysosporium strain C1, strain UV13-6,strain NG7C-19 and strain UV18-25. They have been deposited inaccordance with the Budapest Treaty with the All Russian Collection(VKM) depository institute in Moscow. Wild type C1 strain was depositedin accordance with the Budapest Treaty with the number VKM F-3500 D,deposit date 29-08-1996, C1 UV13-6 mutant was deposited with number VKMF-3632 D, and deposit date Feb. 9, 1998,C1 NG7c-19 mutant was depositedwith number VKM F-3633 D and deposit date Feb. 9, 1998 and C1 UV18-25mutant was deposited with number VKM F-3631 D and deposit date Feb. 9,1998.

[0053] Preferably an expression-regulating region enabling highexpression in the selected host is applied. This can also be a highexpression-regulatingregion derived from a heterologous host, such asare well known in the art. Specific examples of proteins known to beexpressed in large quantities and thus providing suitable expressionregulating sequences for the invention are without being limited theretohydrophobin, protease, amylase, xylanase, pectinase, esterase,beta-galactosidase, cellulase (e.g. endo-glucanase, cellobiohydrolase)and polygalacturonase. The high production has been ascertained in bothsolid state and submerged fermentation conditions. Assays for assessingthe presence or production of such proteins are well known in the art.The catalogues of Sigma and Megazyme for example provide numerousexamples. Megazyme is located at Bray Business Park, Bray, CountyWicklow in Ireland. Sigma Aldrich has many affiliates world wide e.g.USA P.O. Box 14508 St. Louis Mo. For cellulase we refer to commerciallyavailable assays such as CMCase assays, endoviscometric assays,Avicelase assays, beta-glucanase assays, RBBCMCase assays, Cellazyme Cassays. Xylanase assays are also commercially available (e.g. DNS andMegazyme). Alternatives are well known to a person skilled in the artand can be found from general literature concerning the subject and suchinformation is considered incorporated herein by reference. By way ofexample we refer to “Methods in Enzymology Volume 1, 1955 right throughto volumes 297-299 of 1998. Suitably a Chrysosporium promoter sequenceis applied to ensure good recognition thereof by the host.

[0054] We have found that heterologous expression-regulating sequenceswork as efficiently in Chrysosporium as native Chrysosporium sequences.This allows well known constructs and vectors to be used intransformation of Chrysosporium as well as offering numerous otherpossibilities for constructing vectors enabling good rates of expressionin this novel expression and secretion host. For example standardAspergillus transformation techniques can be used as described forexample by Christiansen et al in Bio/Technol. 6:1419-1422 (1988). Otherdocuments providing details of Aspergillus transformation vectors, e.g.U.S. Pat. Nos. 4,816,405, 5,198,345, 5,503,991, 5,364,770 and 5,578,463,EP-B-215.594 (also for Trichoderma) and their contents are incorporatedby reference. As extremely high expression rates for cellulase have beenascertained for Chrysosporiuni strains, the expression regulatingregions of such proteins are particularly preferred. We refer forspecific examples to the previously mentioned deposited Chrysosporiumstrains.

[0055] A nucleic acid construct comprising a nucleic acid expressionregulatory region from Chrysosporium, preferably from Chrysosporiumlucknowense or a derivative thereof forms a separate embodiment of theinvention as does the mutant Chrysosporium strain comprising suchoperably linked to a gene encoding a polypeptide to be expressed.Suitably such a nucleic acid construct will be an expression regulatoryregion from Chrysosporium associated with cellulase or xylanaseexpression, preferably cellobiohydrolase expression, more specificallyexpression of a 55 kDa cellobiohydrolase. The Chrysosporium promotersequences of an endoglucanase of 25 kDa (Cl -EG5) and of anendo-glucanase of 43 kDa (C1-EG6), wherein the molecular weights aredetermined according to SDS PAGE (with the molecular weights accordingto amino acid sequence data being 21.9 kDa and 39.5 kDa), are providedby way of example. Thus, the Chrysosporium promoter sequences ofhydrophobin, protease, amylase, xylanase, esterase, pectinase,beta-galactosidase, cellulase (e.g. endoglucanase, cellobiohydrolase)andpolygalacturonase are considered to also fall within the scope of theinvention. Any of the promoters or regulatory regions of expression ofenzymes disclosed in Table A or B can be suitably employed. The nucleicacid sequence according to the invention can suitably be obtained from aChrysosporium strain according to the invention, such strain beingdefined elsewhere in the description. The manner in which promotersequences can be determined are numerous and well known in the art.Nuclease deletion experiments of the region upstream of the ATG codon atthe beginning of the relevant gene will provide such sequence. Also forexample analysis of consensus sequences can lead to finding a gene ofinterest. Using hybridisation and amplification techniques one skilledin the art can readily arrive at the corresponding promoter sequences.

[0056] The promoter sequences of C1 endoglucanases were identified inthis manner, by cloning the corresponding genes, and are given in SEQ IDNo.'s 5 (EG5) and 6 (EG6), respectively. Other preferred promotersaccording to the invention are the 55 kDa cellobiohydrolase (CBH1)promoter and the 30 kDa xylanase (XylF) promoters, as the enzymes areexpressed at high level by their own promoters. The correspondingpromoter sequences can be identified in a straightforward manner bycloning as described below for the endoglucanase promoters, using thesequence information given in SEQ ID No. 1 (for CBH1) and SEQ ID No. 2(for XylF), respectively. The promoters of thecarbohydrate-degradingenzymes of Chrysosporium, especially C1 promoters,can advantageously be used for expressing desired polypeptides in a hostorganism, especially a fungal or other microbial host organism. Promotersequences having at least 60%, preferably at least 70%, most preferablyat least 80% nucleotide sequence identity with the sequence given in SEQID No's 1 and 2, or with the sequences found for other Chrysosporiumgenes, are part of the present invention. For particular embodiments ofthe recombinant strain and the nucleic acid sequence according to theinvention we also refer to the examples. We also refer for therecombinant strains to prior art describing high expression promotersequences in particular those providing high expression in fungi e.g.such as are disclosed for Aspergillus and Trichoderma. The prior artprovides a number of expression regulating regions for use inAspergillus e.g. U.S. Pat. No. 5,252,726 of Novo and U.S. Pat. No.5,705,358 of Unilever. The contents of such prior art are herebyincorporated by reference.

[0057] The hydrophobin gene is a fungal gene that is highly expressed.It is thus suggested that the promoter sequence of a hydrophobin gene,preferably from Chrysosporium, may be suitably applied as expressionregulating sequence in a suitable embodiment of the invention.Trichoderma reesei and Trichoderma harzianum gene sequences forhydrophobin have been disclosed for example in the prior art as well asa gene sequence for Aspergillus fumigatus and Aspergillus nidulans andthe relevant sequence information is hereby incorporated by reference(Munoz et al, Curr. Genet. 1997, 32(3):225-230; Nakari-Setala T. et al,Eur. J. Biochem. 1996 15:235 (1-2):248-255, M. Partaet al, Infect.Immun. 1994 62 (10): 4389-4395 and Stringer M. A. et al. Mol. Microbiol.1995 16(1):33-44). Using this sequence information a person skilled inthe art can obtain the expression regulating sequences of Chrysosporiumhydrophobin genes without undue experimentation following standardtechniques as suggested already above. A recombinant Chrysosporiumstrain according to the invention can comprise ahydrophobin-regulatingregion operably linked to the sequence encodingthe polypeptide of interest.

[0058] An expression regulating sequence can also additionally comprisean enhancer or silencer. These are also well known in the prior art andare usually located some distance away from the promoter. The expressionregulating sequences can also comprise promoters with activator bindingsites and repressor binding sites. In some cases such sites may also bemodified to eliminate this type of regulation. Filamentous fungalpromoters in which creA sites are present have been described. Such creAsites can be mutated to ensure the glucose repression normally resultingfrom the presence of the non-mutated sites is eliminated. Gist-Brocades'WO 94/13820 illustrates this principle. Use of such a promoter enablesproduction of the polypeptide encoded by the nucleic acid sequenceregulated by the promoter in the presence of glucose. The same principleis also apparent from WO 97/09438. These promoters can be used eitherwith or without their creA sites. Mutants in which the creA sites havebeen mutated can be used as expression regulating sequences in arecombinant strain according to the invention and the nucleic acidsequence it regulates can then be expressed in the presence of glucose.Such Chrysosporium promoters ensure derepression in an analogous mannerto that illustrated in WO 97/09438. The identity of creA sites is knownfrom the prior art. Alternatively, it is possible to apply a promoterwith CreA binding sites that have not been mutated in a host strain witha mutation elsewhere in the repression system e.g. in the creA geneitself, so that the strain can, notwithstanding the presence of creAbinding sites, produce the protein or polypeptide in the presence ofglucose.

[0059] Terminator sequences are also expression-regulating sequences andthese are operably linked to the 3′ terminus of the sequence to beexpressed. Any fungal terminator is likely to be functional in the hostChrysosporium strain according to the invention. Examples are A.nidulans trpC terminator (1), A. niger alpha-glucosidase terminator (2),A. niger glucoamylase terminator (3), Mucor miehei carboxyl proteaseterminator (U.S. Pat. No. 5,578,463) and the Trichoderma reeseicellobiohydrolase terminator. Naturally Chrysosporium terminatorsequences will function in Chrysosporium and are suitable e.g. CBH1 orEG6 terminator.

[0060] A suitable recombinant Chrysosporium strain according to theinvention has the nucleic acid sequence to be expressed operably linkedto a sequence encoding the amino acid sequence defined as signalsequence. A signal sequence is an amino acid sequence which whenoperably linked to the amino acid sequence of the expressed polypeptideallows secretion thereof from the host fungus. Such a signal sequencemay be one normally associated with the heterologous polypeptide or maybe one native to the host. It can also be foreign to both host and thepolypeptide. The nucleic acid sequence encoding the signal sequence mustbe positioned in frame to permit translation of the signal sequence andthe heterologous polypeptide. Any signal sequence capable of permittingsecretion of a polypeptide from a Chrysosporium strain is envisaged.Such a signal sequence is suitably a fungal signal sequence, preferablyan ascomycete signal sequence.

[0061] Suitable examples of signal sequences can be derived from yeastsin general or any of the following specific genera of fungi:Aspergillus, Trichoderma, Chrysosporium, Pichia, Neurospora, Rhizomucor,Hansenula, Humicola, Mucor, Tolypocladium, Fusarium, Penicillium,Saccharomyces, Talaromyces or alternative sexual forms thereof likeEmericella, Hypocrea. Signal sequences that are particularly useful areoften natively associated with the following proteins acellobiohydrolase, an endoglucanase, a beta-galactosidase,a xylanase, apectinase, an esterase, a hydrophobin, a protease or an amylase.Examples include amylase or glucoamylase of Aspergillus or Humicola (4),TAKA amylase of Aspergillus oryzae, alpha-amylase of Aspergillus niger,carboxyl peptidase of Mucor (U.S. Pat. No. 5,578,463), a lipase orproteinase from Rhizomucor miehei, cellobiohydrolase of Trichoderma (5),beta-galactosidaseof Penicillium canescens and alpha mating factor ofSaccharomyces.

[0062] Alternatively the signal sequence can be from an amylase orsubtilisin gene of a strain of Bacillus. A signal sequence from the samegenus as the host strain is extremely suitable as it is most likely tobe specifically adapted to the specific host thus preferably the signalsequence is a signal sequence of Chrysosporium. We have found particularstrains of Chrysosporium to excrete proteins in extremely large amountsand naturally signal sequences from these strains are of particularinterest. These strains are internally designated as Chrysosporiumstrain C1, strain UV13-6, strain NG7C-19 and strain UV18-25. They havebeen deposited in accordance with the Budapest Treaty as describedelsewhere in this description. Signal sequences from filamentous fungi,yeast and bacteria are useful. Signal sequences of non-fungal origin arealso considered useful, particularly bacterial, plant and mammalian.

[0063] A recombinant Chrysosporium strain according to any of theembodiments of the invention can further comprise a selectable marker.Such a selectable marker will permit easy selection of transformed ortransfected cells. A selectable marker often encodes a gene productproviding a specific type of resistance foreign to the non-transformedstrain. This can be resistance to heavy metals, antibiotics and biocidesin general. Prototrophy is also a useful selectable marker of thenon-antibiotic variety. Non-antibiotic selectable markers can bepreferred where the protein or polypeptide of interest is to be used infood or pharmaceuticals with a view to speedier or less complicatedregulatory approval of such a product. Very often the GRAS indication isused for such markers. A number of such markers are available to theperson skilled in the art. The FDA e.g. provides a list of such. Mostcommonly used are selectable markers selected from the group conferringresistance to a drug or relieving a nutritional defect e.g the groupcomprising amdS (acetamidase), hph (hygromycin phosphotransferase), pyrG(orotidine-5′-phosphatedecarboxylase), trpC (anthranilate synthase),argB (ornithine carbamoyltransferase), sC (sulphate adenyltransferase),bar (phosphinothricin acetyl-transferase), glufosinate resistance, niaD(nitrate reductase), a bleomycin resistance gene, more specificallyShble, sulfonylurearesistance e.g. acetolactate synthase mutation ilv 1.Selection can also be carried out by virtue of cotransformation wherethe selection marker is on a separate vector or where the selectionmarker is on the same nucleic acid fragment as the polypeptide-encodingsequence for the polypeptide of interest.

[0064] As used herein the term heterologous polypeptide is a protein orpolypeptide not normally expressed and secreted by the Chrysosporiumhost strain used for expression according to the invention. Thepolypeptide can be of plant or animal (vertebrate or invertebrate)origin e.g. mammalian, fish, insect, or micro-organism origin, with theproviso it does not occur in the host strain. A mammal can include ahuman. A micro-organism comprises viruses, bacteria, archaebacteria andfungi i.e. filamentous fungi and yeasts. Bergey's Manual for BacterialDeterminology provides adequate lists of bacteria and archaebacteria.For pharmaceutical purposes quite often a preference will exist forhuman proteins thus a recombinant host according to the inventionforming a preferred embodiment will be a host wherein the polypeptide isof human origin. For purposes such as food production suitably theheterologous polypeptide will be of animal, plant or algal origin. Suchembodiments are therefore also considered suitable examples of theinvention. Alternative embodiments that are useful also include aheterologous polypeptide of any of bacterial, yeast, viral,archaebacterial and fungal origin. Fungal origin is most preferred.

[0065] A suitable embodiment of the invention will comprise aheterologous nucleic acid sequence with adapted codon usage. Such asequence encodes the native amino acid sequence of the host from whichit is derived, but has a different nucleic acid sequence, i.e. a nucleicacid sequence in which certain codons have been replaced by other codonsencoding the same amino acid but which are more readily used by the hoststrain being used for expression. This can lead to better expression ofthe heterologous nucleic acid sequence. This is common practice to aperson skilled in the art. This adapted codon usage can be carried outon the basis of known codon usage of fungal vis-á-vis non-fungal codonusage. It can also be even more specifically adapted to codon usage ofChrysosporium itself. The similarities are such that codon usage asobserved in Trichoderma, Humicola and Aspergillus should enable exchangeof sequences of such organisms without adaptation of codon usage.Details are available to the skilled person concerning the codon usageof these fungi and are incorporated herein by reference.

[0066] The invention is not restricted to the above mentionedrecombinant Chrysosporium strains, but also covers a recombinantChrysosporium strain comprising a nucleic acid sequence encoding ahomologous protein for a Chrysosporium strain, said nucleic acidsequence being operably linked to an expression-regulatingregion andsaid recombinant strain expressing more of said protein than thecorresponding non-recombinant strain under the same conditions. In thecase of homologous polypeptide of interest such is preferably a neutralor alkaline enzyme like a hydrolase, a protease or a carbohydratedegrading enzyme as already described elsewhere. The polypeptide mayalso be acidic. Preferably the recombinant strain will express thepolypeptide in greater amounts than the non-recombinant strain. Allcomments mentioned vis-A-vis the heterologous polypeptide are also valid(mutatis mutandis) for the homologous polypeptide cellulase.

[0067] Thus the invention also covers genetically engineeredChrysosporium strains wherein the sequence that is introduced can be ofChrysosporium origin. Such a strain can, however, be distinguished fromnatively occurring strains by virtue of for example heterologoussequences being present in the nucleic acid sequence used to transformor transfect the Chrysosporium, by virtue of the fact that multiplecopies of the sequence encoding the polypeptide of interest are presentor by virtue of the fact that these are expressed in an amount exceedingthat of the non-engineered strain under identical conditions or byvirtue of the fact that expression occurs under normally non-expressingconditions. The latter can be the case if an inducible promoterregulates the sequence of interest contrary to the non-recombinantsituation or if another factor induces the expression than is the casein the non-engineered strain. The invention as defined in the precedingembodiments is not intended to cover naturally occurring Chrysosporiumstrains. The invention is directed at strains derived throughengineering either using classical genetic technologies or geneticengineering methodologies.

[0068] All the recombinant strains of the invention can comprise anucleic acid sequence encoding a heterologous protein selected fromcarbohydrate-degrading enzymes (cellulases, xylanases, mannanases,mannosidases, pectinases, amylases, e.g. glucoamylases, -amylases,alpha- and beta-galactosidases, and -glucosidases, -glucanases,chitinases, chitanases), proteases (endoproteases, amino-proteases,amino-and carboxy-peptidases, keratinases), other hydrolases (lipases,esterases, phytases), oxidoreductases (catalases, glucose-oxidases) andtransferases (transglycosylases, transglutaminases, isomerases andinvertases). TABLE A pH range where enzymes retain activity and/orstability pH range retaining > 50% pH range retaining > 70% Stabilityenzymatic activity enzymatic activity (20 h, 50° C.) RBB- Other RBB-Other % from CMC CMC- sub- CMC- CMC sub- max Sample ase ase strates asease strates pH 7.5/8 30 Kd protease (alkaline) 30 kD — — 12.5  — — 12.0 — Xyl (alkaline) — — 10.0  — — 8.5 80 51 kD Xyl — — 8.0 — — 7.5 — 60 kDXyl — — 9.5 — — 9.0 85 45 kD endo 7.0 8.0 — 6.5 7.0 — 75 55 kD endo 8.08.0 — 7.0 7.0 — 55 25 kD (21.8 kD*) endo 7.5 10.0  — 6.5 9.0 — 80 43 kD(39.6 kD*) endo 8.0 8.0 — 7.2 7.2 — — 45 kD, β-Gal/β-Gluc — — 6.8 — —5.7 — 48 kD CBH with β-Gluc traces 5.2 7.5 8.0 5.0 6.8 — — 55 kD CBH 8.09.0 — 7.4 8.5 — 70 65 kD PGU — — 8.0 — — 7.3 — 90 kD protease — — 9.0 —— 9.0 — 100 kD esterase — — 9.0 — — 9.0 —

[0069] TABLE B Activities of enzymes isolated from ultrafiltrate from18-25 strain toward different substrates (pH 5), units/mg protein MUF-RBB- CMC- CMC b-Ghu- pNP-a- pNP-b- Cello- cello- CMC CMC 41 FP (visc)can G G biose Avicel bioside Sample pl 50° C. 40° C. 40° C. 50° C. 40°C. 50° C. 40° C. 40° C. 40° C. 40° C. 40° C. 30 kD procease 8.9 0 0 0 00 0 — 0 0 0 0 30 kD Xyl 9.1 0.1 2 0.1 0.16 0.1 0 — 0 — 0 0 51 kD Xyl 8.70.1 4.2 — 0.19 — 0 — 0 — 0 0 60 kD Xyl 4.7 0 — — 0 — 0 — 0 — 0 0.14 45kD endo 6 51 86 7.6 0.2 47 36 — 0 — 0.5 0 55 kD endo 4.9 47 94 7.7 0.339 25 — 0 — 0.5 0 25 kD (21.8 kD*) endo 4.1 19 15 3.9 0.3 11 3.8 — 0 00.05 0 43 kD (39.6 kD*) endo 4.2 0.43 0.2 0.1 0 0.2 0.2 — 0 0 0 0 45 kDa,b-Gal/b-Gluc 4.2 0 0 0 0 0.01 0.01 0 0.4 0.06 0 0 48 kD CDH withb-Gluc 4.4 0.67 1.3 1.2 0.4 0.8 0.77 0 1.7 0.08 0 0.2 traces +glucono-d- 0 lactone 55 kD CBH 4.4 0.71 0.16 0.27 0.4 0.1 0.1 — 0.050.08 0.46 0.2 with b-Gluc traces + 0 0.14 glucono-d-lactone 65 kD PGU4.4 0 0 0 0 0 0 — 0 0 0 0 90 kD protease 4.2 — — — — — — — — — — — 100kD esterase 4.5 0 0 0 0 0 0 — 0 0 0 0 Polygal- MUF- Gal- pNP-a- pNP-b-MUF- MUF- acturon- gluco- acto- galacto- galacto- Dyed pNP lactosidexyloside Lactose Xylan ic acid side mannan side side casein** butyrateSample 40° C. 40° C. 40° C. 50° C. 50° C. 40° C. 50° C. 40° C. 40° C.50° C. 60° C. 30 kD protease 0 0 0 0 0 0 0 0 0 0.4 0 30 kD Xyl 0 0 — 250 0 0 0 — 0 0 51 kD Xyl 0 0 — 19 0 0 0 0 — 0 0 60 kD Xyl 0.02   0.04 —16.3 0 0 0 0 0 0 0 45 kD endo 0 0 — 1 — 0 1.8 0 — 0 0 55 kD endo 0 0 — 0— 0 0.4 0 — 0 0 25 kD (21.8 kD*) endo 0 — 0 0.03 0 — 0 0 0 0 0 43 kD(39.6 kD*) endo 0 — 0 0 0 — 0 0 0 0 0 45 kD a,b-Gal/b-Gluc 0 —   0.01 0  0.1   0.1   0.2   0.2   0.3 0   1.7 48 kD CDH with b-Gluc 0.36 — 0 0  0.1   0.4 0 0 0 0   2.3 traces + glucono-d- 0.36 lactone 55 kD CBH 0.7— 0 0.1 0 — 0 0 0 0 0 with b-Gluc traces + 0.6 glucono-d-lactone 65 kDPGU 0 — 0 0 1 — 0 0 0 0 0 90 kD protease — — — — — — — — — 0.01 — 100 kDesterase 0 — 0 0 0 0 0 0 0 0 0.8

[0070] The most interesting products to be produced accoprding toinvention are cellulases, xylanases, pectinases, lipases and proteases,wherein cellulases and xylanases cleave beta-1,4-bonds, and cellulasescomprise endoglucanases, cellobiohydrolases and beta-glucosidases. Theseproteins are extremely useful in various industrial processes known inthe art. Specifically for cellulases we refer e.g. to WO 98/15633describing cellobiohydrolases and endoglucanases of use. The contents ofsaid application are hereby incorporated by reference. We also refer toTables A and B providing further details of interesting Chrysosporiumproteins.

[0071] It was found according to the invention, that Chrysosporiummutants can be made that have reduced expression of protease, thusmaking them even more suitable for the production of proteinaceousproducts, especially if the proteinaceous product is sensitive toprotease activity. Thus the invention also involves a mutantChrysosporium strain which produces less protease than non-mutantChrysosporium strain, for example less than C. lucknowense strain C1(VKM F-3500 D). In particular the protease acitivity of such strains isless than half the amount, more in particular less than 30% of theamount produced by C1 strain. The decreased protease activity can bemeasured by known methods, such as by measuring the halo formed op skimmilk plates or BSA degradation.

[0072] An embodiment of the invention that is of particular interest isa recombinant Chrysosporium according to the invention wherein thenucleic acid sequence encoding the polypeptide of interest encodes apolypeptide that is inactivated or unstable at acid pH i.e. pH below 6,even below pH 5,5, more suitably even below pH 5 and even as low as orlower than pH 4. This is a particularly interesting embodiment, as thegenerally disclosed fungal expression systems are not cultured underconditions that are neutral to alkaline, but are cultured at acidic pH.Thus the system according to the invention provides a safe fungalexpression system for proteins or polypeptides that are susceptible tobeing inactivated or are unstable at acid pH.

[0073] Quite specifically a recombinant strain as defined in any of theembodiments according to the invention, wherein the nucleic acidsequence encoding the polypeptide of interest encodes a protein orpolypeptide exhibiting optimal activity and/or stability at a pH above5, preferably at neutral or alkaline pH (i.e. above 7) and/or at a pHhigher than 6, is considered a preferred embodiment of the invention.More than 50%, more than 70% and even more than 90% of optimalactivities at such pH values are anticipated as being particularlyuseful embodiments. A polypeptide expressed under the cultivationconditions does not necessarily have to be active at the cultivationconditions, in fact it can be advantageous for it to be cultured underconditions under which it is inactive as its active form could bedetrimental to the host. This is the case for proteases for example.What is however required is for the protein or polypeptide to be stableunder the cultivation conditions. The stability can be thermalstability. It can also be stability against specific compositions orchemicals, such as are present for example in compositions or processes-of production or application of the polypeptide or protein of interest.LAS in detergent compositions comprising cellulases or lipases, etc. isan example of a chemical often detrimental to proteins. The time periodsof use in applications can vary from short to long exposure so stabilitycan be over a varying length of time varying per application. Theskilled person will be able to ascertain the correct conditions on acase by case basis. One can use a number of commercially availableassays to determine the optimal activities of the various enzymaticproducts. The catalogues of Sigma and Megazyme for example show such.Specific examples of tests are mentioned elsewhere in the description.The manufacturers provide guidance on the application.

[0074] We have surprisingly found that a Chrysosporium strain that canbe suitably used to transform or transfect with the sequence of interestto be expressed is a strain exhibiting relatively low biomass. We havefound that Chrysosporium strains having a biomass two to five timeslower than that of Trichoderma reesei when cultured to a viscosity of200-600 cP at the end of fermentation and exhibiting a biomass of 10 to20 times lower than that of Aspergillus niger when cultured to aviscosity of 1500-2000 cP under corresponding conditions, i.e. theirrespective optimal cultivation conditions can provide a high level ofexpression. This level of expression far exceeds that of the twocommercial reference strains at a much lower biomass and at much lowerviscosity. This means that the yield of expression of such Chrysosporiumstrains will be appreciably higher than from Aspergillus niger andTrichoderma reesei. Such a transformed or transfected Chrysosporiumstrain forms a suitable embodiment of the invention.

[0075] We find a biomass of 0,5- 1,0 g/l for Chrysosporium strain C1(18-25) as opposed to 2,5-5,0 g/l for Trichoderma reesei and 5-10 g/l ofAspergillus niger under the above described conditions. In the Exampleswe provide details of this process.

[0076] In a suitable embodiment a recombinant Chrysosporium strainaccording to the invention produces protein or polypeptide in at leastthe amount equivalent to the production in moles per liter of cellulaseby the strain UV13-6 or C-19, and most preferably at least equivalent toor higher than that of the strain UV18-25 under the corresponding oridentical conditions, i.e. their respective optimal cultivationconditions.

[0077] Unexpectedly we have also found that expression and secretionrates are exceedingly high when using a Chrysosporium strain exhibitingthe mycelial morphology of strain UV18-25 i.e. fragmented short mycelia.Thus a recombinant strain according to the invention will preferablyexhibit such morphology. The invention however also coversnon-recombinant strains or otherwise engineered strains of Chrysosporiumexhibiting this novel and inventive characteristic. Also covered by theinvention is a recombinant Chrysosporium strain in any of theembodiments described according to the invention further exhibitingreduced sporulation in comparison to C1, preferably below that of strainUV13-6, preferably below that of NG7C-19, preferably below that ofUV18-25 under equivalent fermenter conditions. Also covered by theinvention is- a recombinant Chrysosporium strain in any of theembodiments described according to the invention further exhibiting atleast the amount of protein production ratio to biomass in comparison toC1, preferably in comparison to that of any of strains UV13-6, NG7C-19and UV18-25 under equivalent fermenter conditions. The invention howeveralso covers non-recombinant strains or otherwise engineered strains ofChrysosporium exhibiting this novel and inventive characteristic as suchor in combination with any of the other embodiments.

[0078] Another attractive embodiment of the invention also covers arecombinant Chrysosporium strain exhibiting a viscosity below that ofstrain NG7C- 19, preferably below that of UV18-25 under corresponding oridentical fermenter conditions. The invention however also coversnon-recombinant strains or otherwise engineered strains of Chrysosporiumexhibiting this novel and inventive characteristic as such or incombination with any of the other embodiments. We have determined thatthe viscosity of a culture of UV18-25 is below 10 cP opposed to that ofTrichoderma reesei being of the order 200-600 cP, with that ofAspergillus niger being of the order 1500-2000 cP under their respectiveoptimal culture conditions at the end of fermentation. The process usedfor such determination is provided in the examples.

[0079] Viscosity can be assessed in many cases by visual monitoring. Thefluidity of the substance can vary to such a large extent that it can benearly solid, sauce like or liquid. Viscosity can also readily beascertained by Brookfield rotational viscometry, use of kinematicviscosity tubes, falling ball viscometer or cup type viscometer. Theyields from such a low viscosity culture are higher than from thecommercial known higher viscosity cultures per time unit and per cell.

[0080] The processing of such low viscosity cultures according to theinvention is advantageous in particular when the cultures are scaled up.The subject Chrysosporium strains with the low viscosity perform verywell in cultures as large as up to 150,000 liter cultures. Thus anyculture size up to 150,000 litres provides a useful embodiment of theinvention. Any other conventional size of fermentation should be carriedout well with the strains according to the invention. The reasoningbehind this is that problems can arise in large scale production withthe formation of aggregates that have mycelia that are too dense and/orare unevenly distributed. The media as a result cannot be effectivelyutilised during the culture thus leading to an inefficient productionprocess in particular in large scale fermentations i.e. over 150,000liters. Aeration and mixing become problematic leading to oxygen andnutrient starvation and thus reduced concentration of productive biomassand reduced yield of polypeptide during the culture and/or can result inlonger fermentation times. In addition high viscosity and high shear arenot desirable in commercial fermentation processes and in currentcommercial processes they are the production limiting factors. All thesenegative aspects can be overcome by the Chrysosporium host according tothe invention which exhibits much better characteristics thanTrichoderma reesei, Aspergillus niger and Aspergillus oryzae that arecommercially used in this respect i.e. exhibits better proteinproduction levels and viscosity properties and biomass figures.

[0081] A Chrysosporium strain selected from C1, UV13-6, NG7C-19 andUV18-25 illustrates various aspects of the invention exceedingly well.The invention however also covers recombinant strains or otherwiseengineered strains of Chrysosporium derived from the four depositedstrains that also exhibit any of the novel and inventive characteristicsas such or in combination. The deposit data for these strains have beenpresented elsewhere in the description. The invention also coversrecombinant strains or otherwise engineered strains of Chrysosporiumderived from the four deposited strains that also exhibit any of thenovel and inventive characteristics as such or in combination. AChrysosporium strain according to the invention also comprises a strainexhibiting under the corresponding culture conditions a biomass at leasttwice as low as that of Trichoderma reesei, suitably even more up to 5times lower than that of Trichoderma reesei, specifically of aTrichoderma reesei exhibiting a viscosity of 200-600 cP as disclosedunder the conditions of the examples. A Chrysosporium strain accordingto the invention also comprises a strain producing the polypeptide in atleast the amount in moles per liter of cellulase by the strain C1,UV13-6, NG7C-19 or UV18-25 under the corresponding or identicalconditions.

[0082]Chrysosporium strains according to the invention are furtherpreferred if they exhibit optimal growth conditions at neutral toalkaline pH and temperatures of 25-43° C. A preference can exist forneutral and even for alkaline pH. Such production conditions areadvantageous to a number of polypeptides and proteins, in particularthose susceptible to attack by acidic pH or those that are inactive orunstable at low temperatures. It is however also an embodiment of theinvention to include Chrysosporium strains that can be cultured atacidic pH as this can be useful for certain proteins and polypeptides. Asuitable acidic pH lies from 7.0. An acidic pH lower than 6.5 isenvisaged as providing a good embodiment of the invention. A pH around5,0-7,0 is also a suitable embodiment. A neutral pH can be 7.0 or around7 e.g. 6.5-7.5. As stated elsewhere the pH of optimal interest dependson a number of factors that will be apparent to the person skilled inthe art. A pH higher than 7.5 is alkaline, suitably between 7.5-9.0 canbe used.

[0083] When comparing data of strains according to the invention withother strains perhaps having other optimal conditions (e.g. Aspergillusand Trichoderma) for viscosity measurements, biomass determination orprotein production comparisons should be made using the relevant optimalconditions for the relevant strain. This will be obvious to the personskilled in the art.

[0084] A Chrysosporium strain according to any of the above-mentionedembodiments of the invention, said strain further exhibiting productionof one or more of the fungal enzymes selected from thecarbohydrate-degrading enzymes, proteases, other hydrolases,oxidoreductase, and transferases mentioned above is considered aparticularly useful embodiment of the invention. The most interestingproducts are specifically cellulases, xylanases, pectinases, lipases andproteases. Also useful as embodiment of the invention however is aChrysosporium strain exhibiting production of one or more fungal enzymesthat exhibit neutral or alkaline optimal stability and/or activity,preferably alkaline optimal stability and/or activity, said enzyme beingselected from carbohydrate-degrading enzymes, hydrolases and proteases,preferably hydrolases and carbohydrate-degrading enzymes. In the case ofnon-recombinant Chrysosporium, such enzymes are suitably other thancellulase as disclosed in WO 98/15633. Enzymes of particular interestare xylanases, proteases, esterases, alpha galactosidases,beta-galactosidases, beta-glucanases and pectinases. The enzymes are notlimited to the aforementioned. The comments vis-á-vis stability andactivity elsewhere in the description are valid here also.

[0085] The invention also covers a method of producing a polypeptide ofinterest, said method comprising culturing a Chrysosporium strain in anyof the embodiments according to the invention under conditionspermitting expression and preferably secretion of the polypeptide andrecovering the subsequently produced polypeptide of interest.

[0086] Where protein or polypeptide is mentioned, variants and mutantse.g. substitution, insertion or deletion mutants of naturally occurringproteins are intended to be included that exhibit the activity of thenon-mutant. The same is valid vis-á-vis the corresponding nucleic acidsequences. Processes such as gene shuffling, protein engineering anddirected evolution site directed mutagenesis and random mutagenesis areprocesses through which such polypeptides, variants or mutants can beobtained. U.S. Pat. No. 5,223,409, U.S. Pat. No. 5,780,279 and U.S. Pat.No. 5,770,356 provide teaching of directed evolution. Using this processa library of randomly mutated gene sequences created for example by geneshuffling via error prone PCR occurs in any cell type. Each gene has asecretion region and an immobilising region attached to it such that theresulting protein is secreted and stays fixed to the host surface.Subsequently conditions are created that necessitate the biologicalactivity of the particular polypeptide. This occurs for a number ofcycles ultimately leading to a final gene with the desiredcharacteristics. In other words a speeded up directed process ofevolution. U.S. Pat. No. 5,763,192 also describes a process forobtaining DNA, RNA, peptides, polypeptides or protein by way ofsynthetic polynucleotide coupling stochastically generated sequences,introduction thereof into a host followed by selection of the host cellwith the corresponding predetermined characteristic.

[0087] Another application of the method of the present invention is inthe process of “directed evolution”, wherein novel protein-encoding DNAsequences are generated, the encoded proteins are expressed in a hostcell, and those sequences encoding proteins having a desiredcharacteristic are mutated and expressed again. The process is repeatedfor a number of cycles until a protein with the desired characteristicsis obtained. Gene shuffling, protein engineering, error-prone PCR,site-directed mutagenesis, and combinatorial and random mutagenesis areexamples of processes through which novel DNA sequences encodingexogenous proteins can be generated. U.S. Pat. Nos. 5,223,409, 5,780,279and 5,770,356 provide teaching of directed evolution. See also Kuchnerand Arnold, Trends in Biotechnology, 15:523-530 (1997);Schmidt-Dannertand Arnold, Trends in Biotech., 17:135-136 (1999); Arnoldand Volkov, Curr. Opin. Chem. Biol., 3:54-59 (1999); Zhao et al., Manualof Industrial Microbiology and Biotechnology, 2nd Ed., (Demain andDavies, eds.) pp. 597-604, ASM Press, Washington D.C., 1999; Arnold andWintrode, Encyclopedia of Bioprocess Technology: Fermentation,Biocatalysis, and Bioseparation, (Flickinger and Drew, eds.) pp.971-987, John Wiley & Sons, New York, 1999; and Minshull and Stemmer,Curr. Opin. Chem. Biol. 3:284-290.

[0088] An application of combinatorial mutagenesis is disclosed in Hu etal., Biochemistry. 1998 37:10006-10015. U.S. Pat. No. 5,763,192describes a process for obtaining novel protein-encoding DNA sequencesby stochastically generating synthetic sequences, introducing them intoa host, and selecting host cells with the desired characteristic.Methods for effecting artificial gene recombination (DNA shuffling)include random priming recombination (Z. Shao, et al., Nucleic AcidsRes., 26:681 -683 (1998)), the staggered extension process (H. Zhao etal., Nature Biotech., 16:258-262 (1998)), and heteroduplex recombination(A. Volkov et al., Nucleic Acids Res., 27:el 8 (1999)). Error-prone PCRis yet another approach (Song and Rhee, Appl. Environ.Microbiol.66:890-894(2000)).

[0089] There are two widely-practiced methods of carrying out theselection step in a directed evolution process. In one method, theprotein activity of interest is somehow made essential to the survivalof the host cells. For example, if the activity desired is a cellulaseactive at pH 8, a cellulase gene could be mutated and introduced intothe host cells. The transformants are grown with cellulose as the solecarbon source, and the pH raised gradually until only a few survivorsremain. The mutanted cellulase gene from the survivors, which presumablyencodes a cellulase active at relatively high pH, is subjected toanother round of mutation, and the process is repeated untiltransformantsthat can grow on cellulose at pH 8 are obtained.Thermostable variants of enzymes can likewise be evolved, by cycles ofgene mutation and high-temperature culturing of host cells (Liao et al.,Proc. Natl. Acad. Sci. USA 83:576-580(1986); Giver et al., Proc. Natl.Acad. Sci. U S A. 95:12809-12813 (1998).

[0090] An alternative to the massively parallel “survival of thefittest” approach is serial screening. In this approach, individualtransformants are screened by traditional methods, such as observationof cleared or colored zones around colonies growing on indicator media,colorimetric or fluorometric enzyme assays, immunoassays, bindingassays, etc. See for example Joo et al., Nature 399:670-673 (1999),where a cytochrome P450 monooxygenase not requiring NADH as a cofactorwas evolved by cycles of mutation and screening; May et al., NatureBiotech. 18:317-320 (2000), where a hydantoinase of reversedstereoselectivity was evolved in a similar fashion; and Miyazaki et al.,J. Mol. Biol.297:1015-1026(2000), where a thermostable subtilisin wasevolved.

[0091] Standard cloning and protein or polypeptide isolation techniquescan be used to arrive at the required sequence information. Parts ofknown sequences can be used as probes to isolate other homologues inother genera and strains. The nucleic acid sequence encoding aparticular enzyme activity can be used to screen a Chrysosporium libraryfor example. A person skilled in the art will realise whichhybridisation conditions are appropriate. Conventional methods fornucleic acid hybridisation construction of libraries and cloningtechniques are described in Sambrook et al (Eeds) (1989) In “MolecularCloning. A Laboratory Manual” Cold Spring Harbor, Press Plainview, NewYork, and Ausubel et al (Eds) “Current Protocols in Molecular Biology”(1987) John Wiley and Sons, New York. The relevant information can alsobe derived from later handbooks and patents, as well as from variouscommercially available kits in the field.

[0092] In an alternative embodiment, said method comprises culturing astrain according to the invention under conditions permitting expressionand preferably secretion of the protein or polypeptide or precursorthereof and recovering the subsequently produced polypeptide andoptionally subjecting the precursor to additional isolation andpurification steps to obtain the polypeptide of interest. Such a methodmay suitably comprise a cleavage step of the precursor into thepolypeptide or precursor of interest. The cleavage step can be cleavagewith a Kex-2 like protease, any basic amino acid paired protease orKex-2 for example when a protease cleavage site links a well secretedprotein carrier and the polypeptide of interest. A person skilled in theart can readily find Kex-2-like protease sequences as consensus sequencedetails for such are available and a number of alternatives have alreadybeen disclosed e.g. furin.

[0093] Suitably in a method for production of the polypeptide accordingto any of the embodiments of the invention the cultivation occurs at pHhigher than 5, preferably 5-10, more preferably 6-9. Suitably in such amethod the cultivation occurs at a temperature between 25-43 ° C.,preferably 30-40° C. The Chrysosporium strain used in the methodaccording to the invention is quite suitably a recombinant Chrysosporiumstrain according to any of the embodiments disclosed. The methodaccording to the invention in such a case can further be preceded by thestep of production of a recombinant Chrysosporium strain according tothe invention. The selection of the appropriate conditions will dependon the nature of the polypeptide to be expressed and such selection lieswell within the realm of normal activity of a person skilled in the art.

[0094] The method of production of a recombinant Chrysosporium strainaccording to the invention is also part of the subject invention. Themethod comprises stably introducing a nucleic acid sequence encoding aheterologous or homologous polypeptide into a Chrysosporium strain, saidnucleic acid sequence being operably linked to an expression regulatingregion, said introduction occurring in a manner known per se fortransforming filamentous fungi. As stated above numerous referenceshereof are available and a small selection has been cited. Theinformation provided is sufficient to enable the skilled person to carryout the method without undue burden. The method comprises introductionof a nucleic acid sequence comprising any of the nucleic acid elementsdescribed in the various embodiments of the recombinant Chrysosporiumaccording to the invention as such or in combination.

[0095] By way of example the introduction can occur using the protoplasttransformation method. The method is described in the examples.Alternative protoplast or spheroplast transformation methods are knownand can be used as have been described in the prior art for otherfilamentous fungi. Details of such methods can be found in many of thecited references and are thus incorporated by reference. A methodaccording to the invention suitably comprises using a non-recombinantstrain of Chrysosporium according to the invention as starting materialfor introduction of the desired sequence encoding the polypeptide ofinterest.

[0096] The subject invention also covers a method of producingChrysosporium enzyme, said method comprising culturing a Chrysosporiumstrain according to any of the embodiments of the invention as describedabove in or on a cultivation medium at pH higher than 5, preferably5-10, more preferably 6-9, suitably 6-7.5, 7.5-9 as examples of neutraland alkaline pH ranges.

[0097] The subject invention also covers such a method using acultivation medium at a temperature between 25-43° C., preferably 30-40°C. The combination of preferred pH and temperature is an especiallypreferred embodiment of the method of producing Chrysosporium enzymeaccording to the invention.

[0098] More in general the invention further covers a method ofproducing enzymes exhibiting neutral or alkaline optimal activity and/orstability, preferably alkaline optimal activity and/or stability. Thepreferred ranges vis-á-vis pH and optimal activity as well as assayswith which to determine such have been provided elsewhere in thedescription. The enzyme should be selected from carbohydrate-degradingenzymes, proteases, other hydrolases, oxidoreductases, and transferases,as described above, said method comprising cultivating a host celltransformed or transfected with the corresponding enzyme-encodingnucleic acid sequence. Suitably such an enzyme will be a Chrysosporiumenzyme. A suitable method such as this comprises production specificallyof cellulase, xylanase, pectinase, lipase and protease, whereincellulase and xylanase cleave - 1,4-bonds and cellulase comprisesendoglucanase, cellobiohydrolaseand -glucosidase. The method accordingto the invention can comprise cultivating any Chrysosporium hostaccording to the invention comprising nucleic acid encoding suchaforementioned enzymes. Suitably the production of non-recombinantChrysosporium hosts according to the invention is directed at productionof carbohydrate degrading enzymes, hydrolases and proteases. In such acase the enzyme is suitably other than a cellulase. Suitable examples ofproducts to be produced are given in Tables A and B. Methods ofisolating are analogous to those described in WO 98/15633 and areincorporated by reference.

[0099] The enzymes produced by Chrysosporium strains according to theinvention are also covered by the invention. Enzymes of Chrysosporiumorigin as can be isolated from non-recombinant Chrysosporium strainsaccording to the invention are also covered. They exhibit theaforementioned stability, activity characteristics. Suitably they arestable in the presence of LAS. In particular proteases with pI 4-9.5,proteases with a MW of 25-95 kD, xylanases with pI between 4.0 and 9.5,xylanases with MW between 25 and 65 kD, endoglucanases with a pI between3.5 and 6.5, endo-glucanases with MW of 25-55 kDa, B-glucosidases,a,B-galactosidases with a pI of 44.5, β-glucosidases, α,β-galactosidaseswith a MW of 45-50 kDa, cellobiohydrolases of pi 4-5, cellobiohydrolasesof MW 45-75 kDa, e.g. a MW of 55 kD and pI 4.4, polygalacturonases, witha pI of 4.0-5.0 polygalacturonase of 60-70 kDa, e.g. 65 kDa, esteraseswith a pI 4-5, and esterases with a MW of 95-105 kDa with theafore-mentioned stability, activity characteristics are claimed. Themolecular weights (MW) are those determined by SDS-PAGE. Thenon-recombinant i.e. natively occurring enzyme is other than cellulaseas disclosed in WO 98/15633. An enzyme as disclosed in WO 98/15633 isexcluded. Enzymes according to the invention are represented by theenzymes of Table B. Enzymes with combinations of the pI values andmolecular weights mentioned above are also covered.

[0100] The invention is also concerned with the (over)production ofnon-protein products by the mutant (recombinant) strains of theinvention. Such non-protein products include primary metabolites such asorganic acids, amino acids, and secondary such as antibiotics, e.g.penicillins and cephalo-sporins, and other therapeutics. These productsare the result of combinations of biochemical pathways, involvingseveral fungal genes of interest. Fungal primary and secondarymetabolites and procedures for producing these metabolites in fungalorganisms are well known in the art. Examples of the production ofprimary metabolites have been described by Mattey M., The Production ofOrganic Acids, Current Reviews in Biotechnology, 12, 87-132 (1992).Examples of the production of secondary metabolites have been describedby Penalva et al. The Optimization of Penicillin Biosynthesis in Fungi,Trends in Biotechnology 16,483-489(1998).

EXAMPLES

[0101] Examples of Biomass and Viscosity Determinations

[0102] The following operating parameter data ranges have beendetermined for fungal fermentations using three different fungalorganisms. The three fungal organisms compared are: Trichodermalongibrachiatum (formerly T. reesei), Aspergillus niger andChrysosporium lucknowense (UV18-25).

[0103] Viscosity:

[0104] Viscosity is determined on a Brookfield LVF viscometer using thesmall sample adapter and spindle number 31.

[0105] Turn the water-circulating pump on 5 minutes prior to viscometeruse to equilibrate the waterjacket. The water bath temperature should be30° C.

[0106] Obtain a fresh sample of fermentation broth and place 10 ml ofthe broth in the small sample spindle. Select the spindle speed to givea reading in the range 10-80. Wait four (4) minutes and take the readingfrom the viscometer scale. Multiply the reading by the factor givenbelow to get the viscosity in centipoise (cP). Spindle SpeedMultiplication Factor 6 50 12 25 30 10 60 5

[0107] The following viscosity ranges have been determined forfermentations using the specified fungal organism using the aboveprocedure: Viscosity in cP T. longibrachiatum 200-600 A. niger1,500-2,000 C. lucknowense (UV18-25) LT 10

[0108] Biomass:

[0109] Biomass is determined by the following procedure:

[0110] Preweigh 5.5 cm filter paper (Whatman 54) in an aluminiumweighing dish.

[0111] Filter 5.0 ml whole broth through the 5.5 cm paper on a Buchnerfunnel, wash the filter cake with 10 ml deionised water, place thewashed cake and filter in a weighing pan and dry overnight at 60° C.Finish drying at 100° C. for 1 hour, then place in desiccator to cool.

[0112] Measure the weight of dried material. Total biomass (g/l) isequal to the difference between the initial and finals weightsmultiplied by 200.

[0113] The following biomass ranges have been determined forfermentations using the specified fungal organism using the aboveprocedure: Biomass in g/l T. longibrachiatum 2.5-5 A. niger   5-10 C.lucknowense (UV18-25) 0.5-1

[0114] Protein:

[0115] Protein levels were determined using the BioRad Assay Procedurefrom Sigma Company. Protein levels were highest for the Chrysosporium.

[0116] The data presented above represent values determined 48 hoursinto the fermentation process until fermentation end; All values ofAspergilli and Trichoderma are for commercially relevant fungalorganisms and reflect actual commercial data.

[0117] A fungal strain such as C. lucknowense (UV18-25) has theadvantage that the low viscosity permits the use of lower power inputand/or shear the in the fermentation to meet oxygen demands for thosecases where shear stress on the product may be detrimental toproductivity due to physical damage of the product molecule. The lowerbiomass production at high protein production indicates a more efficientorganism in the conversion of fermentation media to product. Thus theChrysosporium provides better biomass and viscosity data whilst alsodelivering at least as much protein, and in fact a lot more protein thanthe two commercially used systems which obviously are better than fortypically deposited Aspergillus or Trichoderma reesei strains in generalpublic collections.

[0118] The high protein production with low biomass concentrationproduced by C. lucknowense (UV18-25) would allow development offermentation conditions with higher multiples of increase in biomass, ifincreasing biomass results in increased productivity, for the desiredproduct before reaching limiting fermentation conditions. The presenthigh levels of biomass and viscosity produced by the T. longibrachiatumand A. niger organisms restrict the increase of biomass as the presentlevels of biomass and viscosity are near limiting practical fermentationconditions.

[0119] Examples of Transformation Comparing Chrysosporium, Trichodermaand Tolypocladium Geodes

[0120] Two untransformed Chrysosporium C1 strains and one Trichodermareesei reference strain were tested on two media (Gs pH 6,8 and Pridhamagar, PA, pH 6,8). To test the antibiotic resistance level spores werecollected from 7 day old PDA plates. Selective plates were incubated at32° C. and scored after 2,4 and 5 days. It followed that the C-1 strainsNG7C-19 and UV18-25 clearly have a low basal resistance level both tophleomycin and hygromycin. This level is comparable to that for areference T. reesei commonly used laboratory strain. Thus there is clearindication these two standard fungal selectable markers can be used wellin Chrysosporium strains. Problems with other standard fungal selectablemarkers should not be expected.

[0121] Selection of Sh-ble (phleomycin-resistance) transformedChrysosporium strains was succesfully carried out at 50 μg/ml. This wasalso the selection level used for T. reesei thus showing thatdifferential selection can be easily achieved in Chrysosporium. The samecomments are valid for transformed strains with hygromycin resistance ata level of 150 μg/ml. TABLE C Gs (pH 6.8) Pridham Agar (PA, pH 6.8)NG7C-19 UV18-25 T.r.11D5 NG7C-19 UV18-25 T.r.11D5 Phleomycin   7.5 μg/ml10 μg/ml 5-7.5 μg/ml 2.5 μg/ml 10-μg/ml 2.5 μg/ml Hygromycin 7.5-10μg/ml 10 μg/ml   10 μg/ml  15 μg/ml  25 μg/ml 15 μg/ml

[0122] The protoplast transformation technique was used on Chrysosporiumbased on the most generally applied fungal transformation technology.All spores from one 90 mm PDA plate were recovered in 8 ml IC1 andtransferred into a shake flask of 50 ml IC1 medium for incubation for 15hours at 35° C. and 200 rpm. After this the culture was centrifuged, thepellet was washed in MnP, brought back into solution in 10 ml MnP and 10mg/ml Caylase C₃ and incubated for 30 minutes at 35° C. with agitation(150 rpm).

[0123] The solution was filtered and the filtrate was subjected tocentrifugation for 10 minutes at 3500 rpm. The pelletwas washed with 10ml MnPCa²⁺. This was centrifugedfor 10 minutes at 25° C. Then 50microlitres of cold MPC was added. The mixture was kept on ice for 30minutes whereupon 2,5 ml PMC was added. After 15 minutes at roomtemperature 500 microlitres of the treated protoplasts were mixed to 3ml of MnR Soft and immediately plated out on a MnR plate containingphleomycin or hygromycin as selection agent. After incubation for fivedays at 30° C. transformants were analysed (clones become visible after48 hours). Transformation efficiency was determined using 10microgrammes of reference plasmid pAN8-1¹⁹. The results are presented inthe following Table D. TABLE D Transformation efficiency (using 10 g ofreference plasmid pAN8-1) T. reesei NG7C-19 UV18-25 Viability 10⁶/200 μl5 10⁶/200 μl 5 10⁶/200 μl Transformants 2500  10⁴  10⁴ Per 200 μlTransformants per 10⁶ 2500 2000 2000 viable cells

[0124] The results show that the Chrysosporium transformants viabilityis superior to that of Trichoderma. The transformability of the strainsis comparable and thus the number of transformants obtained in oneexperiment lies 4 times higher for Chrysosporium than for T. reesei.Thus the Chrysosporium transformation system not only equals thecommonly used T. reesei system, but even outperforms it. Thisimprovement can prove especially useful for vectors that are lesstransformation efficient than pAN8-1. Examples of such less efficienttransformation vectors are protein carrier vectors for production ofnon-fungal proteins which generally yield 10 times fewer transformants.

[0125] A number of other transformation and expression vectors wereconstructed with homologous Chrysosporium protein encoding sequences andalso with heterologous protein encoding sequences for use intransformation experiments with Chrysosporium. The vector maps areprovided in the FIGS. 6-11.

[0126] The homologous protein to be expressed was selected from thegroup of cellulases produced by Chrysosporium and consisted ofendoglucanase 6 which belongs to family 6 (MW 43 kDa) and theheterologous protein was endoglucanase 3 which belongs to family 12 (MW25 kDa) of Penicillium.

[0127] pF6g comprises Chrysosporiwn endoglucanase 6 promoter fragmentlinked to endo-glucanase 6 signal sequence in frame with theendoglucanase 6 open reading frame followed by the endoglucanase6terminator sequence. Transformant selection is carried out by usingcotransformation with a selectable vector.

[0128] pUT1150 comprises Trichoderma reesei cellobiohydrolase promoterlinked to endoglucanase 6 signal sequence in frame with theendoglucanase 6 open reading frame followed by the T reeseicellobiohydrolase terminator sequence. In addition this vector carries asecond expression cassette with a selection marker i.e. the phleomycinresistance gene (Sh-ble gene).

[0129] pUT1152 comprises Aspergillus nidulans glyceraldehyde-3-phosphatedehydrogenase A promoter linked to endoglucanase 6 signal sequence inframe with the endoglucanase 6 open reading frame followed by the A.nidulans anthranilate synthase (trpc) terminator sequence. In additionthis vector carries a second expression cassette with a selection markeri.e. the phleomycin resistance gene (Sh-ble gene).

[0130] pUT1155 comprises A. nidulansglyceraldehyde-3-phosphatedehydrogenase A promoter linked to Trichodermareesei cellobiohydrolase signal sequence in frame with the carrierprotein Sh-ble which in turn is linked in frame to the endoglucanase 6open reading frame followed by the A. nidulans trpC terminator sequence.This vector uses the technology of the carrier protein fused to theprotein of interest which is known to very much improve the secretion ofthe protein of interest.

[0131] pUT1160 comprises Aspergillus nidulans glyceraldehyde-3-phosphatedehydrogenase A promoter linked to Trichoderma reesei cellobiohydrolasesignal sequence in frame with the carrier protein Sh-ble which in turnis linked in frame to the endoglucanase 3 open reading frame ofPenicillium followed by the A. nidulans trpC terminator sequence.

[0132] pUT1162 comprises Trichoderma reesei cellobiohydrolase promoterlinked to endo-glucanase 3 signal sequence in frame with theendoglucanase 3 open reading frame of Penicillium followed by the T.reesei cellobiohydrolase terminator sequence. In addition this vectorcarries a second expression cassette with a selection marker i.e. thephleomycin resistance gene (Sh-ble gene).

[0133] Further examples of expression systems include a Chrysosporiumendoglucanase 3 promoter fragment linked to endoglucanase 3 signalsequence in frame with the endoglucanase3 open reading frame followed bythe endoglucanase 3 terminator sequence. Transformant selection iscarried out by using cotransformation with a selectable vector.

[0134] Another example is a Chrysosporium lucknowense cellobiohydrolasepromoter linked to Penicillium endoglucanase 3 signal sequence in framewith the Penicillium endoglucanase 3 open reading frame followed by theChrysosporium cellobiohydrolase terminator sequence. In addition thisvector carries a second expression cassette with a selection marker i.e.the aceetamidase S gene (AmdS gene).

[0135] A further example comprises Chrysosporiumglyceraldehyde-3-phosphate dehydrogenase 1 promoter linked to theAspergillus niger glucoamylase signal sequence and the glucoamylase openreading frame fused to the human Interleukine 6 open reading frame. Inadddition this vector carries a second expression cassette with aselection marker i.e. the AmdS gene.

[0136] A still further example is a Aspergillus nidulansglyceraldehyde-3-phosphatedehydrogenase A promoter linked to theendoglucanase 5 open reading frame followed by a Aspergillus nidulansterminator sequence. TABLE E Comparative transformations No of Tested inVector Strain Transformation transf. liquid culture PUT1150 UV18-25selection phleo 285 5 T. geodes selection phleo 144 5 PUT1152 UV18-25cotransformationpAN8.1 398 5 T. geodes cotransformationpAN8.1 45 4 PF6gUV18-25 cotransformationpAN8.1 252 6 T. geodes cotransformationpAN8.1127 5 PUT1162 UV18-25 selection phleo >400 T. geodes Not done yet

[0137] Table E shows the results of transformation of both ChrysosporiumUV18-25 and Tolypocladium geodes. The transformation protocol used isdescribed in the section for heterologous transformation.

[0138] Examples of Heterologous and Homologous Expression ofChrysosporium Transformants

[0139] C1 strains (NG7C-19 and/or UV18-25) have been tested for theirability to secrete various heterologous proteins: a bacterial protein(Streptoalloteichus hindustanus phleomycin-resistanceprotein, Sh ble), afungal protein (Trichoderma reesei xylanase II, XYN2) and a humanprotein (the human lysozyme, HLZ).

[0140] The details of the process are as follows:

[0141] [1] C1 secretion of Streptoalloteichushindustanusphleomycin-resistanceprotein (Sh ble).

[0142] C1 strains NG7C-19 and UV18-25 have been transformed by theplasmid pUT720¹. This vector presents the following fungal expressioncassette:

[0143]Aspergillus nidulans glyceraldehyde-3-phosphatedehydrogenase(gpdA) promoter²

[0144] A synthetic Trichoderma reesei cellobiohydrolaseI (cbh1) signalsequence^(1,3)

[0145]Streptoalloteichus hindustanus phleomycin-resistancegene Sh ble ⁴

[0146]Aspergillus nidulans tryptophan-synthase(trpC) terminator⁵

[0147] The vector also carries the beta-lactamase gene (bla) and E. colireplication origin from plasmid pUC18⁶. The detailed plasmid map isprovided in FIG. 2.

[0148] C1 protoplasts were transformed according to Durand et al. ⁷adapted to C1 (media & solutions composition is given elsewhere): Allspores from one 90 mm PDA plate of untransformed C1 strain wererecovered in 8 ml IC1 and transferred into a shake flask with 50 ml IC1medium for incubation 15 hours at 35° C. and 150 rpm. Thereupon, theculture was spun down, the pellet washed in MnP, resolved in 10 mlMnP+10 mg/ml Caylase C₃, and incubated 30 min at 35° C. with agitation(150 rpm). The solution was filtrated and the filtrate was centrifuged10 min at 3500 rpm. The pellet was washed with 10 ml MnPCa²⁺. This wasspun down 10 min at 3500 rpm and the pellet was taken up into 1 mlMnPCa²⁺. 10 μg of pUT720 DNA were added to 200 μl of protoplast solutionand incubated 10 min at room temperature(^(˜)20° C.). Then, 50 μl ofcold MPC was added. The mixture was kept on ice for 30 min whereupon 2.5ml PMC was added. After 15 min at room temperature 500 μl of the treatedprotoplasts were mixed to 3 ml of MnR Soft and immediately plated out ona MnR plate containing phleomycin (50 μg/ml at pH6.5) as selectionagent. After 5 days incubation at 30° C., transformants were analysed(clones start to be visible after 48 hours).

[0149] The Sh ble production of C1 transformants(phleomycin-resistantclones) was analysed as follows: Primarytransformants were toothpicked to GS+phleomycin (5 μg/ml) plates andgrown for 5 days at 32° C. for resistance verification. Each validatedresistant clone was subcloned onto GS plates. Two subclones pertransformant were used to inoculate PDA plates in order to get sporesfor liquid culture initiation. The liquid cultures in IC1 were grown 5days at 27° C. (shaking 200 rpm). Then, the cultures were centrifuged(5000 g, 10 min.) and 500 μl of supernatant were collected. From thesesamples, the proteins were precipitated with TCA and resuspended inWestern Sample Buffer to 4 mg/ml of total proteins (Lowry Method ⁸). 10μl (about 40 μg of total proteins) were loaded on a 12% acrylamide/SDSgel and run (BioRad Mini Trans-Blot system). Western blotting wasconducted according to BioRad instructions (Schleicher & Schull 0.2 μmmembrane) using rabbit anti-Sh ble antiserum (Cayla Cat. Ref.#ANTI-0010) as primary antibody.

[0150] The results are shown in FIG. 1 and Table F: TABLE F Sh bleestimated production levels in C1 Estimated Sh ble Estimated Sb blequantity on the concentration in the Western blot production mediaUntransformedNG7C-19 Not detectable NG7C-19::720clone 4-1  25 ng 0.25mg/l NG7C-19::720clone 5-1  25 ng 0.25 mg/l NG7C-19::720clone 2-2 250 ng 2.5 mg/l Untransformed UV18-25 Not detectable UV18-25::720clone 1-2 500ng   5 mg/l UV18-25::720clone 3-1 250 ng  2.5 mg/l

[0151] These data show that:

[0152] 1) The heterologous transcription/translation signals from pUT720are functional in Chrysosporium.

[0153] 2) The heterologous signal sequence of pUT720 is functional inChrysosporium.

[0154] 3) Chrysosporium can be used a host for the secretion of anheterologous bacterial protein.

[0155] [2] C1 secretion of the human lysozyme (HLZ).

[0156] C1 strains NG7C-19 and UV18-25 have been transformed by theplasmid pUT970G ⁹. This vector presents the following fungal expressioncassette:

[0157]Aspergillus nidulans glyceraldehyde-3-phosphatedehydrogenase(gpdA) promoter²

[0158] A synthetic Trichoderma reesei cellobiohydrolaseI (cbh1) signalsequence ^(1,3)

[0159]Streptoalloteichus hindustanus phleomycin-resistancegene Sh ble ⁴used as carrier-protein ¹⁰

[0160]Aspergillus niger glucoamylase (glaA2) hinge domain cloned fromplasmid pAN56-2 ^(11,12)

[0161] A linker peptide (LGERK) featuring a KEX2-like protease cleavagesite ¹

[0162] A synthetic human lysozyme gene (hlz) ¹⁰

[0163]Aspergillus nidulans tryptophan-synthase(trpC) terminator ⁵

[0164] The vector also carries the beta-lactamase gene (bla) and E. colireplication origin from plasmid pUC18 ⁶. The detailed plasmid map isprovided in FIG. 3. C1 protoplasts were transformed with plasmid pUT970Gfollowing the same procedure already described in example 1. The fusionprotein (Sh ble :: GAM hinge :: HLZ) is functional with respect to thephleomycin-resistance thus allowing easy selection of the C1transformants. Moreover, the level of phleomycin resistance correlatesroughly with the level of hlz expression.

[0165] The HLZ production of C1 transformants (phleomycin-resistantclones) was analysed by lysozyme-activity assay as follow: Primarytransformants were toothpicked to GS+phleomycin (5μg/ml) plates(resistance verification) and also on LYSO plates (HLZ activitydetection by clearing zone visualisation ^(1, 10)) Plates were grown for5 days at 32° C. Each validated clone was subcloned onto LYSO plates.Two subclones per transformant were used to inoculate PDA plates inorder to get spores for liquid culture initiation. The liquid culturesin IC1 were grown 5 days at 27° C. (shaking 180 rpm). Then, the cultureswere centrifuged (5000 g, 10 min.). From these samples, lysozymeactivity was measured according to Morsky et al. ¹³. TABLE G Active HLZproduction levels in C1 Active HLZ concentration in culture mediaUntransformedNG7C-19 0 mg/l NG7C-19::970G clone 4 4 mg/l NG7C-19::970Gclone 5 11 mg/l UntransformedUV18-25 0 mg/l UV18-25::970G clone 1 8 mg/lUV18-25::970G clone 2 4 mg/l UV18-25::970G clone 3 2 mg/l UV18-25::970Gclone 2 2.5 mg/l

[0166] These data show that:

[0167] 1) Points 1 & 2 from example 1 are confirmed.

[0168] 2) Sh ble is functional in Chrysosporium as resistance-marker.

[0169] 3) Sh ble is functional in Chrysosporium as carrier-protein.

[0170] 4) The KEX2-like protease cleavage site is functional inChrysosporium (otherwise HLZ wouldn't be active).

[0171] 5) Chrysosporium can be used as host for the secretion of aheterologous mammalian protein.

[0172] [3] C1 secretion of Trichoderma reesei xylanase II (XYN2).

[0173] C1 strain UV18-25 has been transformed by the plasmids pUT1064and pUT1065. pUT1064 presents the two following fungal expressioncassettes:

[0174] The first cassette allows the selection ofphleomycin-resistanttransformants:

[0175]Neurospora crassa cross-pathway control gene 1 (cpc-1) promoter¹⁴

[0176]Streptoalloteichus hindustanus phleomycin-resistancegene Sh ble ⁴

[0177]Aspergillus nidulans tryptophan-synthase(trpC) terminator⁵

[0178] The second cassette is the xylanase production cassette:

[0179]T. reesei_strain TR2 cbh1 promoter¹⁵

[0180]T. reesei_strain TR2 xyn2 gene (including its signal sequence)16

[0181]T. reesei_strain TR2 cbh1 terminator ¹⁵

[0182] The vector also carries an E. coli replication origin fromplasmid pUC19 ⁶. The plasmid detailed map is provided in FIG. 4.

[0183] pUT1065 presents the following fungal expression cassette:

[0184]A. nidulans glyceraldehyde-3-phosphatedehydrogenase(gpdA)promoter²

[0185] A. synthetic T. _(—) reesei cellobiohydrolasel (cbh1) signalsequence ^(1,3)

[0186]S. hindustanus phleomycin-resistancegene Sh ble ⁴ used ascarrier-protein¹⁰

[0187] A linker peptide (SGERK) featuring a KEX2-like protease cleavagesite ¹

[0188]T. reesei_strain TR2xyn2 gene (without signal sequence)¹⁶

[0189]A. nidulans tryptophan-synthase(trpC) terminator⁵

[0190] The vector also carries the beta-lactamase gene (bla) and an E.coli replication origin from plasmid pUC18 ⁶. The plasmid detailed mapis provided in FIG. 5. C1 protoplasts were transformed with plasmidpUT1064 or pUT1065 following the same procedure already described inexample 1. The fusion protein in plasmid pUT1065 (Sh ble :: XYN2) isfunctional with respect to the phleomycin-resistance thus allowing easyselection of the C1 transformants. Moreover, the level of phleomycinresistance correlates roughly with the level of xyn2 expression. InpUT1064, xyn2 was cloned with its own signal sequence.

[0191] The xylanase production of C1 transformants (phleomycin-resistantclones) was analysed by xylanase-activity assay as follow: Primarytransformants were toothpicked to GS+phleomycin (5 μg/ml) plates(resistance verification) and also on XYLAN plates (xylanase activitydetection by clearing zone visualisation ¹⁷). Plates were grown for 5days at 32° C. Each validated clone was subcloned onto XYLAN plates. Twosubclones per transformant were used to inoculate PDA plates in order toget spores for liquid culture initiation. The liquid cultures in IC1+5g/1 KPhtalate were grown 5 days at 27° C. (shaking 180 rpm). Then, thecultures were centrifuged (5000 g, 10 min.). From these samples,xylanase activity was measured by DNS Technique according to Miller etal. ¹⁸ TABLE H Active XYN2 production levels in C1 (best producers)Active xylanase II Xylanase II specific concentration in activity inculture media culture media Untransformed UV 18-25  3.9 U./ml  3.8 U./mgtotal prot. UV18-25::1064 clone 7-1  4.7 U./ml  4.7 U./mg total prot.UV18-25::1064 clone 7-2  4.4 U./ml  4.3 U./mg total prot. UV18-25::1065clone 1-1 29.7 U./ml 25.6 U./mg total prot. UV18-25::1065 clone 1-2 30.8U./ml 39.4 U./mg total prot.

[0192] These data show that:

[0193] 1) Points 1 to 4 from example 2 are confirmed.

[0194] 2) C1 can be used as host for the secretion of a heterologousfungal protein.

[0195] [4] We also illustrate data from expression of transformedUV18-25 wherin the table 1 shows the results for the plasmids with whichtransformation was carried out. The Table shows good expression levelsfor endoglucanase and cellobiohydrolase using heterologous expressionregulating sequences and signal sequences but also with homologousexpression regulating sequences and signal sequences. The details of thevarious plasmids can be derived elsewhere in the description and fromthe figures. The production occurs at alkaline pH at a temperature of35° C. TABLE I Expression data of transformed UV18-25 strain Totalproteins CMCase β-glucanase Culture mg/ml u/ml u/mg u/ml u/mg pH value*UV 100% 100% 100% 100% 100% 7.90  18-25 1150-23  94% 105% 111% 140%149% 7.90   -30  96% 105% 110% 145% 151% 8.10 1152-3  94% 112% 120% 147%156% 7.85   -4 100% 105% 105% 132% 132% 7.90 1160-2  69%  81% 118%  90%131% 7.90   -4  73%  72%  98%  83% 114% 8.35   -1  92%  95% 103% 120%130% 8.45 1162-1 102% 105% 103% 145% 142% 8.20   -11 112% 109%  98% 115%103% 8.20  F6g-20 104% 102%  98% 130% 125% 7.90   -25 — — — — — —

[0196] Appendixto the Examples: Media

[0197] Transformation Media: Mandels Base: MnP Medium: KH₂PO₄ 2.0 g/lMandels Base with (NH₄)₂SO₄ 1.4 g/l Peptone 1 g/l MgSO₄.7H₂O 0.3 g/l MES2 g/l CaCl₂ 0.3 g/l Sucrose 100 g/l Oligoelements 1.0 ml/l Adjust pH to5 MnR MnP CA²⁺: MnP + sucrose 130 g/l MnP Medium + 50 mM Yeast extract2.5 g/l CaCl₂ 2H₂O Glucose 2.5 g/l Adjust pH to 6.5 Agar 15 g/l MnRSoft: MnR with only 7.5 g/l of agar. MPC: CaCl₂ 50 mM pH 5.8 MOPS 10 mMPEG 40%

[0198] For Selection and Culture GS: Glucose 10 g/l [Merieux] Biosoyase 5 g/l pH should be 6.8 Agar 15 g/l PDA: Potato Dextrose Agar 39 g/l[Difco] pH should be 5.5 MPG: Mandels Base with K. Phtalate  5 g/lGlucose 30 g/l Yeast extract  5 g/l

[0199] The regeneration media (MnR) supplemented with 50 g/ml phleomycinor 100-150 μg/ml hygromycin is used to select transformants. GS medium,supplemented with 5 μg/ml phleomycin is used to confirm antibioticresistance.

[0200] PDA is a complete medium for fast growth and good sporulation.Liquid media are inoculated with {fraction (1/20)}th of spore suspension(all spores from one 90 mm PDA plate in 5 ml 0.1% Tween). Such culturesare grown at 27° C. in shake flasks (200 rpm).

[0201] Isolation and Characterisation of C1 Proteins

[0202] The process for obtaining various proteins is described as are anumber of characteristics of the proteins. The tables A, B and J providedetails of purification scheme and activities. Isolation occurs from theChrysosporium culture filtrate using DEAE-Toyopearl ion exchangechromatography analogously to the method described in WO 98/15633, whichis incorporated herein by reference. The non-bound fraction (F 60-31 CF)obtained from this chromatography was purified using Macro Prep Q ionexchange chromatography after equilibration to pH 7,6. The non-boundfraction (NBNB) was pooled and bound proteins were eluted in 0-1 M NaClgradient. The NBNB fraction provided major protein bands of 19, 30, 35and 46 kD and a minor one of 51 kD. In 0-1 M NaCl gradient protein peakswere eluted from various fractions. 39-41 included 28, 36 and 60 kDproteins, 44-48 included 28, 45 and 66 kD as major protein bands with33, 36, 55, 60 and 67 kD proteins, the 49-51 fraction gave 30, 36, 56and 68 kD proteins and the 52-59 fraction included major 33 and 55 kDproteins and minor 28 and 36 kD proteins. The pooled NBNB fraction wasfurther purified by hydrophobic chromatographyon Phenyl Superose. TheNBNB fraction was equilibrated with 0,03M Na-phosphate buffer pH 7,0containing 1,2 M (NH₄)₂SO₄ and applied to a column. Adsorbed proteinswere eluted in 1,2-0,6 M (NH₄)₂SO₄ gradient. Thus homogeneous xylanasewith MW 30 and 51 kD and pI 9.1 and 8.7 respectively were obtained aswas a 3 0 kD protease with pI 8,9.

[0203] The xylanases did not possess MUF cellobiase activity and arethus true xylanases. The alkaline 30 kD xylanase (pI 9.1) possessed highactivity within a very broad pH range from 5-8 maintaining 65% ofmaximum activity at pH 9- 10; it is a member of the xylanase F family;its partial nucleotide and amino acid sequences are depicted in SEQ IDNo. 7. The partial amino acid sequence depicted corresponds to aboutamino acids 50-170 from the N terminus of the mature protein. Xylanasesaccording to invention have at least 60%, preferably at least 70%, mostpreferably at least 80% sequence identity of the partial amino acidsequence of SEQ ID No. 7. The corresponding xylanase promoter, which isa preferred embodiment of the invention, can be identified using thepartial nucleotide sequence of SEQ ID No. 7. The 51 kD xylanase (pI 8,7)possessed maximum activity at pH 6 and retained at least 70% of itsactivity at pH 7,5 and it retained at least 50% of its activity at pH8,0. It was not very stable with only 15% activity at pH 5,5 and 4% atpH 7,5. The Michaelis constant toward birch xylan was 4,2 g/l for 3OkDxylanase and 3,4 g/l for 51 kD xylanase. Temperature optimum was highand equal to 70° C. for both xylanases.

[0204] The 30 kD protease activity measured towards proteins of the NBNBfraction appeared to be equal to 0,4×10³ units/ml at 50° C. and pH 7,90kD. The fraction exhibited activity toward dyed casein of 0,4 arbitraryunits/mg (pH 7). Addition of urea as chaotropic agent resulted in 2-3times increase of protease activity. The effect of the protease onxylanase activity was significant. Only 30% xylanase activity remainedat pH 10,3 and 50° C. after 30 minutes of incubation. At pH 8 95% of thexylanase activity remained. LAS addition resulted in a dramatic decreaseof xylanase activity at pH 8 and 10,3 with only 50% xylanase activityafter 10 minutes of incubation with or without protease inhibitor PMSF.The 30 kD protease was alkaline with pH optimum at pH 10-11. Theactivity is inhibited by phenylmethylsulfonyl fluoride (PMSF) and not byiodoacetic acid, pepstatin A and EDTA which characterises it as a serinetype protease. The protease is not active towards C1 proteins at neutralpH and 50° C. without chaotropic agents. Increase of pH and the additionof chaotropic agents such as LAS, SDS and urea significantly increaseproteolysis.

[0205] The 39-41 fraction was purified by hydrophobic chromatography onplenol superose. Fractions were equilibrated with 0,03M Na phosphatebuffer pH 7,2 containing 1,5 M (NH₄)₂SO₄ and applied to a column.Adsorbed proteins were eluted in 1,5-0 M (NH₄)₂SO₄ gradient. Thushomogenous xylanase with MW 60 kD and pI 4,7 was obtained. This xylanasepossessed activities towards xylan, MUF-cellobioside, MUF-xyloside andMUF-lactoside. This xylanase probably belongs to family 10 (family F).This xylanase was stable at pH from 5 to 8 during 24 hours and retainedmore than 80% activity at 50° C. It retained 70% activity at pH 5-7 at60° C. It kept 80% activity during 5 hours and 35% during 24 hours at50° C. and pH 9. At pH 10 60% activity was retained at 50° C. and 0,5hours of incubation. After 5 hours of incubation at pH 8 and 60° C. 45%activity was found decreasing to 0 after 24 hours. It had a pH optimumwithin the pH range of 6-7 and kept 70% activity at pH 9 and 50% of itsactivity at pH 9,5. The Michaelis constant toward birch xylan was 0,5g/l. Temperature optimum was high and equal to 80° C.

[0206] Fraction 44-48 was then purified by chromatofocusing on Mono P. ApH gradient from 7,63-5,96 was used for the elution of the proteins. Asa result 45 kD endoglucanase was isolated with a pI of 6. The 45 kD endohad maximum activity at pH 5 toward CMC and at pH 5-7 toward RBB-CMC.The 45 kD endo retained 70% of its maximal activity toward CMC at pH 6,5and 70% of its maximal activity toward RBB-CMC was retained at pH 7,0;50% of its maximal activity toward CMC was retained at pH 7 and 50% ofits maximal activity toward RBB-CMC was retained at pH 8. The Michaelisconstant toward CMC was 4,8 g/l. Temperature optimum was high and equalto 80° C. Other proteins 28, 33, 36, 55, 60 and 66 kD were eluted mixedtogether.

[0207] Fraction 52-58 was purified by chromatofocusing on Mono P toowith a pH gradient 7,6-4,5. Individual 55 kD endoglucanase with pI 4,9was obtained. The 55 kD endo was neutral. It has a broad pH optimum from4,5-6 and 70% activity was retained at pH 7,0 both for CMC and RBB-CMCand 50% activity was retained at pH 8 for both CMC and RBB-CMC. TheMichaelis constant toward CMC was 1 g/l. Temperature optimum was highand equal to 80° C. A number of fractions also held proteins with MW of28,33 and 36 kD.

[0208] 45, 48 and 100 kD proteins were isolated from bound DEAEToyopearl fraction of F 60-8 UF conc of Chrysosporium culture fromfractions 50-53 using Macro Prep Q chromatography.

[0209] Fraction 50-53 was equilibrated with 0.03 M imidazole HCL buffer,pH 5.75 and was applied to a column and the adsorbed proteins wereeluted in 0,1 -0,25 M NaCl gradient for 4 h. As a result 45 kD (pI4.2),48 kD (pI 4.4) and 100 kD (pI 4.5) proteins were isolated inhomogenous states.

[0210] The 45 kD is supposedly a alpha beta-galactosidase by virtue ofits activity toward p-nitrophenyl alpha-galactoside and p-nitrophenylbeta-galactoside. The pH optimum was 4,5 70% activity was maintained atpH 5,7 and 50% of its activity was retained at pH 6,8. The temperatureoptimum was 60° C.

[0211] The 48 kD protein was a cellobiohydrolase having high activitytoward p-nitrophenyl beta-glucoside and also activities toward MUFcellobioside, MUF lactoside and p-nitrophenyl butyrate. The 48 kDprotein had a pH optimum of S toward CMC and 5-6 toward RBB-CMC.

[0212] The 100 kD protein with pI 4,5 possessed activity only towardp-nitrophenyl butyrate. It is probably an esterase but is not a feruloylesterase as it had no activity against methyl ester of ferulic acid. Ithad neutral/alkalinepH optimum (pH 8-9) and optimal temperature of55-60° C.

[0213] The 90 kD protease with pI 4,2 was isolated from the boundfraction and the activity measured towards proteins of the NBNB fractionappeared to be equal to 12×10⁻³ units/ml at 50° C. and pH 7,90 kD. Thefraction exhibited activity toward dyed casein of 0,01 arbitraryunits/mg (pH 7). Addition of urea as chaotropic agent resulted in 2-3fold increase of protease activity as did addition of LAS at both pH 7and 9 (50° C.). The 90 kD protease was neutral with pH optimum at pH 8.The activity is inhibited by phenylmethylsulfonyl fluoride (PMSF) andnot by iodoacetic acid, pepstatin A and EDTA which characterises it as aserine type protease.

[0214] Also isolated from the bound fraction were 43 kD endoglucanasewith pl 4.2 (fraction 33-37) and 25 kD endoglucanase with pI 4.1(fraction 39-43), 55 kD cellobiohydrolase with pI 4.9 (fraction 39-43)and 65 kD polygalacturonase with pI 4.4 (fraction 39-43). Theendoglucanases did not possess activity towards avicel or MUFcellobioside and possessed high activity toward MC, RBB-CMC, CMC41,beta-glucan and endoglucanase. The 25 kD endo did not produce glucosefrom CMC and the 43 kD endo did. No glucose was formed from avicel. ThepH optimum for the 43 kD protein was 4,5 with 70% maximum activitymaintained at pH 7.2 and 50% at pH 8. The 43 kD endo kept 70% activityat pH 5 and 6 during 25 hours of incubation. It kept only 10% at pH 7during this incubation period. The 25 kD endo had pH optimum of activityat pH 5 toward CMC and broad pH optimum of activity toward RBB-CMC with70% of the maximum activity being kept at pH 9 and with 50% of themaximum activity being at pH 10. It kept 100% activity at pH 5 and 6 and80% at pH 7, 8, 8.6 and 9.6 during 120 hours of incubation. The 25 kDendo had a temperature optimum of activity at 70° C. The 43 kD endo hada temperature optimum of activity at 60° C. The Michaelis constantstowards CMC were 62 and 12,7 g/l for 25 and 43 kD endo respectively. Thepoly-galacturonase is a pectinase. The Michaelis constant toward PGA was3.8 g/l. The pH optimum of PGU activity is within pH range 5-7 and Toptimum within 50-65° C.

[0215] Genes encoding C. lucknowense proteins were obtained using PCRand characterised by sequence analysis. The corresponding full geneswere obtained by screening (partial) gene libraries using the isolatedPCR fragments. The full gene of the 43 kD endoglucanase (EG6, Family 6)of the C1 strain has been cloned, sequenced and analysed (including 2.5kb promoter region and 0.5 kb terminator region). Its nucleotide andamino acid sequences are depicted in SEQ ID No.6. Predicted molecularweight of the mature protein is 39,427 Da and predicted pI is 4.53,which values correspond well with the measured values. Protein alignmentanalysis with other glycosyl hydrolases of the family 6.2 shows thatC1-EG6 does not include a cellulose-binding domain (CBD) Homologyanalysis using SwissProt SAMBA software (Smith & Waterman algorithm, Gappenalty 12/2, alignment 10, Blosum62 matrix) shows that C1-EG6 has 51.6%identity with Fusarium oxysporum EG-B (over 376 amino acids), 51.0%identity with Agaricus bisporus CBH3 (over 353 amino acids), and 50.7%identity with Trichoderma reesei CBH2 (over 367 amino acids). Theputative signal sequence runs Met 1 to Arg 28. The promoter containsseveral potential CreA binding sites, so it is very likely that thispromoter would be subject to glucose repression in a fungal strain withworking CreA regulation.

[0216] Similarly, the full gene of the 25 kD endoglucanase (EG5, Family45) of the C1 strain has been cloned, sequenced and analysed (including3.3 kb promoter region and 0.7 kb terminator region). The nucleotide andamino acid sequences are depicted in SEQ ID No. 5. Predicted molecularweight of the mature protein is 21,858 Da and predicted pl is 4.66,which values correspond well with the measured values. This is thesmallest fungal endoglucanase known to date. Protein alignment analysiswith other glycosyl hydrolases of the family 45 shows that C1-EG5 doesnot include a cellulose-binding domain (CBD), nor a cohesin/dockerindomain. Homology analysis using NCBI-BLASTP2 software (Gap penalty 11/1,alignment 10, Blosum62 matrix) shows that the closest homologous proteinto C1-EG5 is Fusarium oxysporum EG-K with 63% identity. The putativesignal sequence runs Met 1 to Ala 18. The promoter contains manypotential CreA binding sites, so it is very likely that this promoterwould be subject to glucose repression in a fungal strain with workingCreA regulation.

[0217] Furthermore, an additional endoglucanase was found by PCR basedon family 12 cellulases homology analysis. The partial nucleotide andamino acid sequence of this additional endoglucanase (EG3, Family 12) isgiven in SEQ ID No. 8.

[0218] The 55kD protein was a cellobiohydrolase (referred to herein asCBH1) with activity against MUF-cellobioside, MUF lactoside, FP andavicel, also against p-nitrophenyl -glucoside, cellobiose andp-nitrophenyl lactoside. Its activity toward MUF cellobioside isinhibited by cellobiose. The inhibition constant 0,4 mM was determined.The Michaelis constant toward MUF cellobioside was 0,14 mM, toward MUFlactoside was 4 mM and toward CMC was 3,6 g/l. The pH optimum is ratherbroad from 4,5 to 7. 50% of maximum activity toward CMC and 80% activitytoward RBB-CMC is kept at pH 8. 70-80% activity within pH 5-8 is keptduring 25 hours of incubation. The temperature optimum is 60-70° C. CBH1is probably a member of the cellobiohydrolase family 7; its partialnucleotide and amino acid sequences are depicted in SEQ ID No. 9. Thepartial amino acid sequence depicted corresponds to about amino acids300-450 from the N terminus of the mature protein. A cellobiohydrolaseaccording to the invention has at least 60%, preferably at least 70%,most preferably at least 80% sequence identity of the partial amino acidsequence of SEQ ID No. 9. The corresponding CBH promoter, which is apreferred embodiment of the invention, can be identified using thepartial nucleotide sequence of SEQ ID No. 9. A synergistic effect wasobserved between 25 kD endo and 55 kD CBH during avicel hydrolysis.Synergism coefficient was maximal at the ratio of 25 kD endo to 55 kDCBH 80:20. The K_(syn) was 1,3 at its maximum.

[0219] Tables A, B and J illusrate the details of the above. TABLE JPurification scheme Purification of F 60-8 (UF-conc) amd F 60-31 CFsamples.

[0220] The expression level of five main Chrysosporium genes was studiedby Northern analysis. Various strains of C. lucknowense were grown inrich medium containing pharmedia with cellulose and lactose (medium 1)or rich medium containing pharnedia and glucose (medium 2) at 33 C.After 48 h, mycelium was harvested and RNA was isolated. The RNA washybridised with 5 different probes: EG5, EG6, EG3, XyIF and CBH. Afterexposure, the Northern blots were stripped and hybridised again with aprobe for ribosomal L3 as a control for the amount of mRNA on the blot.Most strains showed very high response for CBH and high response forXylF in medium 1; in medium 2, half of the strain showed high responsefor all genes, and the other half showed low response. The order ofexpression strength was deducted from these data asCBH>XyIF>EG5>EG3>EG6.

[0221] Tables A, B and J illustrate the details of the above.

[0222] Advanced Isolation and Characterisation of C1 Genes and GeneExpression Sequences of CBH1, XYL1 . EG3 and GPD

[0223] Construction of a BlueSTAR gene library of UV18-25

[0224] Chromosomal DNA of UV18-25 was partially digested with Sau3A,fragments of 12-15 kb were isolated and ligated in a BamHI site ofcloning vector BlueSTAR. Packaging of 20% of the ligation mixtureresulted in a gene library of 4.6×10⁴ independent clones. This librarywas multiplied and stored at 4° C. and −80° C. The rest of the ligationmixture was also stored at 4° C. Screening the gene library ofUV18-25for isolation of the genes for cbh1, eg3, xyl1 and gpd1 For theisolation of the different genes, in total ±7.5×10⁴ individual BlueSTARphages per probe were hybridized in duplo. Hybridisation was carried outwith the PCR fragments of cbh1, eg3 and xyl1 (as described inPCT/NL99/00618)at homologous conditions (65° C.; 0.2xSSC) and with thegpd1 gene of A. niger at heterologous conditions (53° C.; 0.5xSSC). Thenumber of positive signals is given in Table K. The positive clones wererescreened and for each clone two individual phages were used forfurther experiments. DNA of the different clones was analysed byrestriction analysis to determine the number of different clonesisolated from each gene (results are given in Table K).

[0225] As for each of the 4 genes, 4-6 different clones were isolated,we conclude that the primary gene library (±4-5×10⁴ clones) representsabout 5× genome of UV 1 8-25. From this result we conclude that thecomplete genome of UV 18-25 is represented in 9×10³ clones. Based on anaverage genomic insert of 13 kb, this would indicate a genome size of±120 Mb, which is 3 times the size of the Aspergillus genome.

[0226] PCR reactions with specific primers for the gene present on theplasmid (based on previous sequence determination from the isolated PCRfragments) and the T7 and T3 primer present in the polylinker ofpBlueSTAR we were able to determine the location of the genes in anumber of clones. From each gene a plasmid was used for sequencedetermination of the gene.

[0227] Sequence analysis of the clonedgenes

[0228] For the cbh1, xyl1, eg3 and the gpd1 gene, the results of thesequence determination are represented in SEQ ID No's 1, 2, 3 and 4respectively. Also the deduced amino acid sequences of the proteins arerepresented in these SEQ ID No's 1-4. Some properties of the proteinsare given in Table L. It should be mentioned that the position of thestart of the translation and the introns is based on homology with genesfrom the same family (i.e. paper genetics).

[0229] CBH1

[0230] From the amino acid sequences of CBH1, we concluded that theprotein is about 63 kD in size and that a cellulose binding domain (CBD)is present at the C-terminal part of the protein. Interestingly, noevidence was found for the presence of a CBD in the isolated 55 kD majorprotein. However, the presence of the isolated peptides from this 55 kDmajor protein in the encoded CBHI protein (SEQ ID No.1), confirms thatthe 55 kD protein is encoded by the cloned gene. A possible explanationof these results is that the 55 kD protein is a truncated version of theCBH1 protein lacking the CBD.

[0231] Xyl1

[0232] From the amino acid sequences of xyl1 we conclude that also herea CBD is present, in this protein at the N-terminal side. In theliterature only two more xylanases with a CBD are known (Fusariumoxysporum and Neocallimastix patriciarum). The estimated size of theXyl1 protein is 43 kD and several peptides isolated from a 30 kDxylanase originate from this protein (SEQ ID No. 2). It should be notedthat a considerable number of the isolated peptides could not be foundin the encoded sequence. This could indicate that alternative xylanaseproteins are present in UV18-25. In previous analysis, no evidence wasfound for the presence of CBD in this 30 kD protein. Also from theseresults we hypothesized that the CBD of the protein is cleaved of byproteolysis. This hypothesis will be analysed further (by determinationof activities, N-terminal sequences and sizes of the different proteinsin the different C1 strains: C1 wild type, NG7C, UV13-6, UV18-25 andprotease mutants of UV18-25 ) Also the effect of the presence or absenceof the CBD on enzymatic activities has to be analysed in detail further.Overexpression of the full length genes in various C1 hosts may beconsidered. The presence of a cellulose binding domain (CBD) is aparticular feature of this enzyme; the only other known family 10glycolytic enzyme (xylanase) having a CBD is the Fusarium oxysporumXylF. The invention thus pertains to fungal xylanases having a CBD otherthan the Fusarium oxysporumxylanase.

[0233] EG3

[0234] From the amino acid sequence of EG3 it could be concluded thatEG3 is a family 12 protein. The gene encodes a preproprotein with adibasic (K-R) propeptide processing site. The C1EG3 protein is 62%similar and 54% identical to the B1 EG3 protein. One putativeglycosylation site is present at the C-terminal part of the protein (SEQID No. 3).

[0235] Gpd1

[0236] The DNA sequence of the C-terminal part of the gpd1 gene is notdetermined, since we are primarily interested in the promoter sequencesof this gene (SEQ ID No.4).

[0237] The proteins XYL1 and EG3 of C. lucknowense are 54-70% identicalto their closest homologue in the Genbank DATABASE (Table L). Notable isthe strong homology of the CBH1 and the EG5 proteins to their relatedHumicola grisea proteins (74-82% identical). Interestingly the closestrelated proteins to the EG6 protein are only 46-48% identical.

[0238] Also notable is that in most cases the closest homologuesoriginate from Fusarium, Humicola or other Pyrenomycetous fungi (TableL), whereas Chrysosporium belongs to the Plectomycetous fungi accordingto the NCBI taxonomy database (Table L). TABLE K Screening of 7.5 × 10⁴phages of the gene library of UV18-25 with PCR fragments of UV 18-25 forthe cbh1 gene, the eg3 gene and the xy11 gene (homologous conditions)and with the gpdA gene of A. niger (heterologous conditions). DNAisolation and restriction analysis was used to determine the number ofdifferent clones. Positive in positive in clone used for Gene firstscreening rescreening different clones sequencing cbh1 8 7 4 pCBH7 eg3 66 4 pEG3-3 xyl1 9 6 5 pXyl5 gpd1 12 12 6 pGPD4

[0239] TABLE L isolated number glycosidase from of amino relatedsequences family C1 acids introns remarks (% identity/% homology) CBH1 7 70 kD 526 1 CBD Humicola grisea (74/82) 55 kD (63 kD) (CBH1: P15828)Fusarium oxysporum (58/68) (CBH: P46238) Neurospora crassa (60/69)(CBH1: P38676) XYL1 10 30 kD 333 3 CBD Fusarium oxysporum (67/72) (43kD) (XylF: P46239) Penicillium simplissicum (63/72) (XylF: P56588)Aspergillus aculeatus (61/70) (XylF: O59859) EG3 12 — 247 2 preproAspergillus aculeatus (60/71) (30 kD + peptide (F1-CMCase: P22669)glycos) Hypocrea jecorina (56/73) (EG: BAA20140) Aspergillus kawachii(54/69) (CMCase: Q12679) EG6 6(2) 43 kD 395 2 no CBD Fusarium oxysporum(48/59) (EGLB: P46236) Acremonium cellulolyticus (48/58) (CBHII:BAA74458) Agaricus bisporus (46/59) (CBH3: P49075) EG5 45 25 kD 225 3 noCBD Humicola grisea (82/91) (EG: BAA74957) Fusarium oxysporum (63/78)(EGL-K: P45699) Humicola grisea (62/78) (EG: BAA74956) GPD1 — — In- 2+?— Podospora anserina (85/89) complete (GPD: P32637) Neurospora crassa80/86) (GPD: U67457) Cryphonectria parasitica 80/85) (GPD: P19089)

DESCRIPTION OF THE FIGURES

[0240]FIG. 1 is a Western blot as described in the Examples

[0241]FIG. 2 is a pUT720 map

[0242]FIG. 3 is a pUT970G map

[0243]FIG. 4 is a pUT1064 map

[0244]FIG. 5 is a pUT1065 map

[0245]FIG. 6 is a pF6g map

[0246]FIG. 7 is a pUT1150 map

[0247]FIG. 8 is a pUT1152 map

[0248]FIG. 9 is a pUT1155 map

[0249]FIG. 10 is a pUT1160 map

[0250]FIG. 11 is a pUT1162 map

[0251]FIG. 12: Ion exchange chromatographyon Macro Prep Q of NB-fractionafter DEAE-Toyopearlof F-60-31 CF sample.

[0252]FIG. 13: pH courses of activities of 30kD (pl 8.9) and 90 kD (pl4.2) proteases toward C1 proteins (50° C., 30 min. incubation).

[0253]FIG. 14: Effect of 30 kD (pI 8.9) “alkaline” protease on xylanaseactivity of the NBNB-fraction (Macro Prep Q) of F 60-31 CF at 50°.

[0254]FIG. 15: Effect of 90 kD (pI 4.2)“neutral” protease on CMCaseactivity of the proteins in the bound fraction #44-45 (DEAE-Toyopearl)ofF 60-8 UV-conc sample at 50° C.

[0255]FIG. 16: Complete hydrolysis of polygalacturonicacid by 65 kDpolygalacturonase(pl 4.4): 50° C., pH 4.5; concentration pf PGA=5 g/l,concentration of protein=0.1 g/l.

[0256]FIG. 17: pH- and temperature dependencies ofpolygalacturonaseactivity of F-60-43 UF-conc.

[0257]FIG. 18: Inhibition of activity toward MUF-cellobiosidebycellobiose for 55 kD CBH (pI 4.4): pH 4.5, 40° C.

[0258]FIG. 19: Synergistic effect between 25 kD Endo (pI 4.1) and 55 kDCBH (pI 4.4) toward avicel (40° C., pH 5, 25 min).

[0259]FIG. 20: Complete hydrolysis of CMC (a) and avicel (b) by theenzymes isolated from bound fractions of F-60-8 UF-conc. sample (50° C.,pH 5): concentration of CMC and avicel=5 g/l, concentration of 25 kDEndo=0.01 g/l, concentration of 43 kD Endo=0.02 g/l; 1-25 kD Endo (pI4.1),2-43 kD Endo (pI 4.2).

[0260]FIG. 21: Complete hydrolysis of CMC (1) and avicel (2) by 55 kDCBH (pI 4.4) without (a) and with (b) glucono- -lactone(50° C., pH 4.5):concentration of CMC and avicel=5 g/l, concentrationof protein=0.1 g/l,concentration of glucono- -lactone=5 g/l.

[0261]FIG. 22: pH-Dependence is of CMCase and RBB-CMCase activities ofthe enzymes isolated from F-60-8 UF-conc. sample: 1-25 kD Endo (pI4.1),2-43 kD Endo (pI 4.2).

[0262]FIG. 23: pH-Dependencies of CMCase and RBB-CMCase activities of 55kD CBH (pI 4.4).

[0263]FIG. 24: Temperature dependencies of CMCase activity (pH 4.5) ofthe enzymes isolated from bound fractions of F-60-8 UF-conc. sample:1-55 kD CBH (pI 4.4),2-25 kD Endo (pI 4.1), 3-43 kD Endo (pI 4.2).

[0264]FIG. 25: pH-stability(50° C.) of the enzymes isolated from boundfractions of F-60-8 UF-conc. sample: 1-55 kD CBH (pI 4.4),2-25 kD Endo(pI 4.1),3-43 kD Endo (pI 4.2).

[0265]FIG. 26: Adsorption of the enzymes isolated from bound fractionsof F-60-8 UF-conc. sample.

[0266] References (The contents hereof are incorporated)

[0267] 1. Calmels T. P., Martin F., Durand H., and Tiraby G. (1991)Proteolytic events in the processing of secreted proteins in fungi. JBiotechnol 17(1): p.51-66.

[0268] 2. Punt P. J., Dingemanse M. A., Jacobs-Meijsing B. J., PouwelsP. H., and van den Hondel C. A. (1988) Isolation and characterization ofthe glyceraldehyde-3-phosphatedehydrogenase gene of Aspergillusnidulans. Gene 69(1): p. 49-57.

[0269] 3. Shoemaker S., Schweickart V., Ladner M., Gelfand D., Kwok S.,Myambo K., and Innis M. (1983) Molecular cloning ofexo-cellobiohydrolasel derived from Trichoderma reesei strain L27.Bio/Technology Oct.:691-696.

[0270] 4. Drocourt D., Calmels T., Reynes J. P., Baron M., and Tiraby G.(1990) Cassettes of the Streptoalloteichus hindustanus ble gene fortransformation of lower and higher eukaryotes to phleomycin resistance.Nucleic Acids Res 18(13): p.4009.

[0271] 5. Mullaney E. J., Hamer J. E., Roberti K. A., Yelton M. M., andTimberlake W. E. (1985) Primary structure of the trpC gene fromAspergillus nidulans. Mol Gen Genet 199(1): p.37-45.

[0272] 6. Yanisch-Perron C., Vieira J., and Messing J. (1987) ImprovedM13 phage cloning vectors and host strains: nucleotide sequences of theM13mp18 and pUC19 vectors. Gene 33:103-119.

[0273] 7. Durand H., Baron M., Calmels T., and Tiraby G. (1988)Classical and molecular genetics applied to Trichodermareesei for theselection of improved cellulolytic industrial strains, in Biochemistryand genetics of cellulose degradation, J. P. Aubert, Editor. AcademicPress. p. 135-151.

[0274] 8. Lowry O. H., Rosebrough N. J., Farr A. L., and Randall R. J.(1951) Protein measurements with the folin phenol reagent. J. Biol.Chem. ?: 193-265.

[0275] 9. Parriche M., Bousson J. C., Baron M., and Tiraby G.Development of heterologous protein secretion systems in filamentousfungi. in 3rd European Conference on Fungal Genetics. 1996. Münster,Germany.

[0276] 10. Baron M., Tiraby G., Calmels T., Parriche M., and Durand H.(1992) Efficient secretion of human lysozyme fused to the Sh blephleomycin resistance protein by the fungus Tolypocladium geodes. JBiotechnol24(3): p. 253-266.

[0277] 11. Jeenes D. J., Marczinke B., MacKenzie D. A., and Archer D. B.(1993) A truncated glucoamylase gene fusion for heterologous proteinsecretion from Aspergillus niger. FEMS Microbiol. Lett. 107(2-3): p.267-271.

[0278] 12. Stone P. J., Makoff A. J., Parish J. H., and Radford A.(1993) Cloning and sequence-analysis of the glucoamylase gene ofneurospora-crassa. Current Genetics 24(3): p. 205-211.

[0279] 13. Morsky P. (1983) Turbidimetric determination of lysozyme withMicrococcus lysodeikticus cells: Reexamination of reaction conditions.Analytical Biochem. 128:77-85.

[0280] 14. Paluh J. L., Orbach M. J., Legerton T. L., and Yanofsky C.(1988) The cross-pathway control gene of Neurospora crassa, cpc-1,encodes a protein similar to GCN4 of yeast and the DNA-binding domain ofthe oncogene v-jun-encoded protein. Proc Natl Acad Sci U S A 85(11): p.3728-32.

[0281] 15. Nakari T., Onnela M. L., Ilmen M., Nevalainen K., andPenttilä M. (1994) Fungal promoters active in the presence of glucose,Patent #WO 94/04673, Alko.

[0282] 16. Torronen A., Mach R. L., Messner R., Gonzalez R., KalkkinenN., Harkki A., and Kubicek C. P. (1992) The two major xylanasesfromTrichodermareesei: characterization of both enzymes andgenes.Biotechnology(N Y) 10(11): p.1461-5.

[0283] 17. Farkas V. (1985) Novel media for detection of microbialproducers of cellulase and xylanase. FEMS Microbiol. Letters 28:137-140.

[0284] 18. Miller G. L. (1959) Use of dinitrosalicylic acid reagent fordetermination of reducing sugar. Anal. Chem. 31:426-428.

[0285] 1. 19.Punt P. J., Mattern I. E., van den Hondel C.A.M.J.J. (1988)A vector for Aspergillus transformation conferring phleomycinresistance. Fungal Genetics Newsletter 35, 25-30.

1 12 1 1570 DNA Homo sapiens CDS (126)..(1523) 1 gtcgacgttg caggctgagtcatcactaga gagtgggaag ggcagcagca gcagagaatc 60 caaaccctaa agctgatatcacaaagtacc atttctccaa gttgggggct cagaggggag 120 tcatc atg agc gat gttacc att gtg aaa gaa ggt tgg gtt cag aag agg 170 Met Ser Asp Val Thr IleVal Lys Glu Gly Trp Val Gln Lys Arg 1 5 10 15 gga gaa tat ata aaa aactgg agg cca aga tac ttc ctt ttg aag aca 218 Gly Glu Tyr Ile Lys Asn TrpArg Pro Arg Tyr Phe Leu Leu Lys Thr 20 25 30 gat ggc tca ttc ata gga tataaa gag aaa cct caa gat gtg gat tta 266 Asp Gly Ser Phe Ile Gly Tyr LysGlu Lys Pro Gln Asp Val Asp Leu 35 40 45 cct tat ccc ctc aac aac ttt tcagtg gca aaa tgc cag tta atg aaa 314 Pro Tyr Pro Leu Asn Asn Phe Ser ValAla Lys Cys Gln Leu Met Lys 50 55 60 aca gaa cga cca aag cca aac aca tttata atc aga tgt ctc cag tgg 362 Thr Glu Arg Pro Lys Pro Asn Thr Phe IleIle Arg Cys Leu Gln Trp 65 70 75 act act gtt ata gag aga aca ttt cat gtagat act cca gag gaa agg 410 Thr Thr Val Ile Glu Arg Thr Phe His Val AspThr Pro Glu Glu Arg 80 85 90 95 gaa gaa tgg aca gaa gct atc cag gct gtagca gac aga ctg cag agg 458 Glu Glu Trp Thr Glu Ala Ile Gln Ala Val AlaAsp Arg Leu Gln Arg 100 105 110 caa gaa gag gag aga atg aat tgt agt ccaact tca caa att gat aat 506 Gln Glu Glu Glu Arg Met Asn Cys Ser Pro ThrSer Gln Ile Asp Asn 115 120 125 ata gga gag gaa gag atg gat gcc tct acaacc cat cat aaa aga aag 554 Ile Gly Glu Glu Glu Met Asp Ala Ser Thr ThrHis His Lys Arg Lys 130 135 140 aca atg aat gat ttt gac tat ttg aaa ctacta ggt aaa ggc act ttt 602 Thr Met Asn Asp Phe Asp Tyr Leu Lys Leu LeuGly Lys Gly Thr Phe 145 150 155 ggg aaa gtt att ttg gtt cga gag aag gcaagt gga aaa tac tat gct 650 Gly Lys Val Ile Leu Val Arg Glu Lys Ala SerGly Lys Tyr Tyr Ala 160 165 170 175 atg aag att ctg aag aaa gaa gtc attatt gca aag gat gaa gtg gca 698 Met Lys Ile Leu Lys Lys Glu Val Ile IleAla Lys Asp Glu Val Ala 180 185 190 cac act cta act gaa agc aga gta ttaaag aac act aga cat ccc ttt 746 His Thr Leu Thr Glu Ser Arg Val Leu LysAsn Thr Arg His Pro Phe 195 200 205 tta aca tcc ttg aaa tat tcc ttc cagaca aaa gac cgt ttg tgt ttt 794 Leu Thr Ser Leu Lys Tyr Ser Phe Gln ThrLys Asp Arg Leu Cys Phe 210 215 220 gtg atg gaa tat gtt aat ggg ggc gagctg ttt ttc cat ttg tcg aga 842 Val Met Glu Tyr Val Asn Gly Gly Glu LeuPhe Phe His Leu Ser Arg 225 230 235 gag cgg gtg ttc tct gag gac cgc acacgt ttc tat ggt gca gaa att 890 Glu Arg Val Phe Ser Glu Asp Arg Thr ArgPhe Tyr Gly Ala Glu Ile 240 245 250 255 gtc tct gcc ttg gac tat cta cattcc gga aag att gtg tac cgt gat 938 Val Ser Ala Leu Asp Tyr Leu His SerGly Lys Ile Val Tyr Arg Asp 260 265 270 ctc aag ttg gag aat cta atg ctggac aaa gat ggc cac ata aaa att 986 Leu Lys Leu Glu Asn Leu Met Leu AspLys Asp Gly His Ile Lys Ile 275 280 285 aca gat ttt gga ctt tgc aaa gaaggg atc aca gat gca gcc acc atg 1034 Thr Asp Phe Gly Leu Cys Lys Glu GlyIle Thr Asp Ala Ala Thr Met 290 295 300 aag aca ttc tgt ggc act cca gaatat ctg gca cca gag gtg tta gaa 1082 Lys Thr Phe Cys Gly Thr Pro Glu TyrLeu Ala Pro Glu Val Leu Glu 305 310 315 gat aat gac tat ggc cga gca gtagac tgg tgg ggc cta ggg gtt gtc 1130 Asp Asn Asp Tyr Gly Arg Ala Val AspTrp Trp Gly Leu Gly Val Val 320 325 330 335 atg tat gaa atg atg tgt gggagg tta cct ttc tac aac cag gac cat 1178 Met Tyr Glu Met Met Cys Gly ArgLeu Pro Phe Tyr Asn Gln Asp His 340 345 350 gag aaa ctt ttt gaa tta atatta atg gaa gac att aaa ttt cct cga 1226 Glu Lys Leu Phe Glu Leu Ile LeuMet Glu Asp Ile Lys Phe Pro Arg 355 360 365 aca ctc tct tca gat gca aaatca ttg ctt tca ggg ctc ttg ata aag 1274 Thr Leu Ser Ser Asp Ala Lys SerLeu Leu Ser Gly Leu Leu Ile Lys 370 375 380 gat cca aat aaa cgc ctt ggtgga gga cca gat gat gca aaa gaa att 1322 Asp Pro Asn Lys Arg Leu Gly GlyGly Pro Asp Asp Ala Lys Glu Ile 385 390 395 atg aga cac agt ttc ttc tctgga gta aac tgg caa gat gta tat gat 1370 Met Arg His Ser Phe Phe Ser GlyVal Asn Trp Gln Asp Val Tyr Asp 400 405 410 415 aaa aag ctt gta cct cctttt aaa cct caa gta aca tct gag aca gat 1418 Lys Lys Leu Val Pro Pro PheLys Pro Gln Val Thr Ser Glu Thr Asp 420 425 430 act aga tat ttt gat gaagaa ttt aca gct cag act att aca ata aca 1466 Thr Arg Tyr Phe Asp Glu GluPhe Thr Ala Gln Thr Ile Thr Ile Thr 435 440 445 cca cct gaa aaa tgt cagcaa tca gat tgt ggc atg ctg ggt aac tgg 1514 Pro Pro Glu Lys Cys Gln GlnSer Asp Cys Gly Met Leu Gly Asn Trp 450 455 460 aaa aaa taa taaaaagtaagtttcaatag ctaaaaaaaa aaaaaaaaaa aaaaaaa 1570 Lys Lys 465 2 465 PRT Homosapiens 2 Met Ser Asp Val Thr Ile Val Lys Glu Gly Trp Val Gln Lys ArgGly 1 5 10 15 Glu Tyr Ile Lys Asn Trp Arg Pro Arg Tyr Phe Leu Leu LysThr Asp 20 25 30 Gly Ser Phe Ile Gly Tyr Lys Glu Lys Pro Gln Asp Val AspLeu Pro 35 40 45 Tyr Pro Leu Asn Asn Phe Ser Val Ala Lys Cys Gln Leu MetLys Thr 50 55 60 Glu Arg Pro Lys Pro Asn Thr Phe Ile Ile Arg Cys Leu GlnTrp Thr 65 70 75 80 Thr Val Ile Glu Arg Thr Phe His Val Asp Thr Pro GluGlu Arg Glu 85 90 95 Glu Trp Thr Glu Ala Ile Gln Ala Val Ala Asp Arg LeuGln Arg Gln 100 105 110 Glu Glu Glu Arg Met Asn Cys Ser Pro Thr Ser GlnIle Asp Asn Ile 115 120 125 Gly Glu Glu Glu Met Asp Ala Ser Thr Thr HisHis Lys Arg Lys Thr 130 135 140 Met Asn Asp Phe Asp Tyr Leu Lys Leu LeuGly Lys Gly Thr Phe Gly 145 150 155 160 Lys Val Ile Leu Val Arg Glu LysAla Ser Gly Lys Tyr Tyr Ala Met 165 170 175 Lys Ile Leu Lys Lys Glu ValIle Ile Ala Lys Asp Glu Val Ala His 180 185 190 Thr Leu Thr Glu Ser ArgVal Leu Lys Asn Thr Arg His Pro Phe Leu 195 200 205 Thr Ser Leu Lys TyrSer Phe Gln Thr Lys Asp Arg Leu Cys Phe Val 210 215 220 Met Glu Tyr ValAsn Gly Gly Glu Leu Phe Phe His Leu Ser Arg Glu 225 230 235 240 Arg ValPhe Ser Glu Asp Arg Thr Arg Phe Tyr Gly Ala Glu Ile Val 245 250 255 SerAla Leu Asp Tyr Leu His Ser Gly Lys Ile Val Tyr Arg Asp Leu 260 265 270Lys Leu Glu Asn Leu Met Leu Asp Lys Asp Gly His Ile Lys Ile Thr 275 280285 Asp Phe Gly Leu Cys Lys Glu Gly Ile Thr Asp Ala Ala Thr Met Lys 290295 300 Thr Phe Cys Gly Thr Pro Glu Tyr Leu Ala Pro Glu Val Leu Glu Asp305 310 315 320 Asn Asp Tyr Gly Arg Ala Val Asp Trp Trp Gly Leu Gly ValVal Met 325 330 335 Tyr Glu Met Met Cys Gly Arg Leu Pro Phe Tyr Asn GlnAsp His Glu 340 345 350 Lys Leu Phe Glu Leu Ile Leu Met Glu Asp Ile LysPhe Pro Arg Thr 355 360 365 Leu Ser Ser Asp Ala Lys Ser Leu Leu Ser GlyLeu Leu Ile Lys Asp 370 375 380 Pro Asn Lys Arg Leu Gly Gly Gly Pro AspAsp Ala Lys Glu Ile Met 385 390 395 400 Arg His Ser Phe Phe Ser Gly ValAsn Trp Gln Asp Val Tyr Asp Lys 405 410 415 Lys Leu Val Pro Pro Phe LysPro Gln Val Thr Ser Glu Thr Asp Thr 420 425 430 Arg Tyr Phe Asp Glu GluPhe Thr Ala Gln Thr Ile Thr Ile Thr Pro 435 440 445 Pro Glu Lys Cys GlnGln Ser Asp Cys Gly Met Leu Gly Asn Trp Lys 450 455 460 Lys 465 3 24 DNAArtificial Sequence Description of Artificial SequenceSynthethicOligonucleotide Primers 3 tccaaaccct aaagctgata tcac 24 4 22 DNAArtificial Sequence Description of Artificial SequenceSyntheticOligonucleotide Primers 4 cctggatagc ttctgtccat tc 22 5 74 DNAArtificial Sequence Description of Artificial SequenceSyntheticOligonucleotide Primers 5 atgagcgatg ttaccattgt gaaagaaggt tgggttcagaagaggggaga atatataaaa 60 aactggaggc caag 74 6 27 DNA Artificial SequenceDescription of Artificial SequenceSynthetic Oligonucleotide Primers 6ttattttttc caggtaccca gcatgcc 27 7 90 DNA Artificial SequenceDescription of Artificial SequenceSynthetic Oligonucleotide Primers 7gcgcgcgaat tcccaccatg ggtagcaaca agagcaagcc caaggatgcc agccagcggc 60gccgcagcag cgatgttacc attgtgaaag 90 8 66 DNA Artificial SequenceDescription of Artificial SequenceSynthetic Oligonucleotide Primers 8gcgcgcgggc ccttaggcgt agtcggggac gtcgtacggg tattttttcc agttacccag 60catgcc 66 9 51 DNA Artificial Sequence Description of ArtificialSequenceSynthetic Oligonucleotide Primers 9 cggggtacca ccatgggtagcaacaagagc aagcccaagg atgccagcca g 51 10 30 DNA Artificial SequenceDescription of Artificial SequenceSynthetic Oligonucleotide Primers 10ccggaattct taggcgtagt cggggacgtc 30 11 480 PRT Homo sapiens 11 Met AsnGlu Val Ser Val Ile Lys Glu Gly Trp Leu His Lys Arg Gly 1 5 10 15 GluTyr Ile Lys Thr Trp Arg Pro Arg Tyr Phe Leu Leu Lys Ser Asp 20 25 30 GlySer Phe Ile Gly Tyr Lys Glu Arg Pro Glu Ala Pro Asp Gln Thr 35 40 45 LeuPro Pro Leu Asn Asn Phe Ser Val Ala Glu Cys Gln Leu Met Lys 50 55 60 ThrGlu Arg Pro Arg Pro Asn Thr Phe Val Ile Arg Cys Leu Gln Trp 65 70 75 80Thr Thr Val Ile Glu Arg Thr Phe His Val Asp Ser Pro Asp Glu Arg 85 90 95Glu Glu Trp Met Arg Ala Ile Gln Met Val Ala Asn Ser Leu Lys Gln 100 105110 Arg Ala Pro Gly Glu Asp Pro Met Asp Tyr Lys Cys Gly Ser Pro Ser 115120 125 Asp Ser Ser Thr Thr Glu Glu Met Glu Val Ala Val Ser Lys Ala Arg130 135 140 Ala Lys Val Thr Met Asn Asp Phe Asp Tyr Leu Lys Leu Leu GlyLys 145 150 155 160 Gly Thr Phe Gly Lys Val Ile Leu Val Arg Glu Lys AlaThr Gly Arg 165 170 175 Tyr Tyr Ala Met Lys Ile Leu Arg Lys Glu Val IleIle Ala Lys Asp 180 185 190 Glu Val Ala His Thr Val Thr Glu Ser Arg ValLeu Gln Asn Thr Arg 195 200 205 His Pro Phe Leu Thr Ala Leu Lys Tyr AlaPhe Gln Thr His Asp Arg 210 215 220 Leu Cys Phe Val Met Glu Tyr Ala AsnGly Gly Glu Leu Phe Phe His 225 230 235 240 Leu Ser Arg Glu Arg Val PheThr Glu Glu Arg Ala Arg Phe Tyr Gly 245 250 255 Ala Glu Ile Val Ser AlaLeu Glu Tyr Leu His Ser Arg Asp Val Val 260 265 270 Tyr Arg Asp Ile LysLeu Glu Asn Leu Met Leu Asp Lys Asp Gly His 275 280 285 Ile Lys Ile ThrAsp Phe Gly Leu Cys Lys Glu Gly Ile Ser Asp Gly 290 295 300 Ala Thr MetLys Thr Phe Cys Gly Thr Pro Glu Tyr Leu Ala Pro Glu 305 310 315 320 ValLeu Glu Asp Asn Asp Tyr Gly Arg Ala Val Asp Trp Trp Gly Leu 325 330 335Gly Val Val Met Tyr Glu Met Met Cys Gly Arg Leu Pro Phe Tyr Asn 340 345350 Gln Asp His Glu Arg Leu Phe Glu Leu Ile Leu Met Glu Glu Ile Arg 355360 365 Phe Pro Arg Thr Leu Ser Pro Glu Ala Lys Ser Leu Leu Ala Gly Leu370 375 380 Leu Lys Lys Asp Pro Lys Gln Arg Leu Gly Gly Gly Pro Ser AspAla 385 390 395 400 Lys Glu Val Met Glu His Arg Phe Phe Leu Ser Ile AsnTrp Gln Asp 405 410 415 Val Val Gln Lys Lys Leu Leu Pro Pro Phe Lys ProGln Val Thr Ser 420 425 430 Glu Val Asp Thr Arg Tyr Phe Asp Asp Glu PheThr Ala Gln Ser Ile 435 440 445 Thr Ile Thr Pro Pro Asp Arg Tyr Asp SerLeu Gly Leu Leu Glu Leu 450 455 460 Asp Gln Arg Thr His Phe Pro Gln PheSer Tyr Ser Ala Ser Ile Arg 465 470 475 480 12 465 PRT Homo sapiens 12Met Ser Asp Val Thr Ile Val Lys Glu Gly Trp Val Gln Lys Arg Gly 1 5 1015 Glu Tyr Ile Lys Asn Trp Arg Pro Arg Tyr Phe Leu Leu Lys Thr Asp 20 2530 Gly Ser Phe Ile Gly Tyr Lys Glu Lys Pro Gln Asp Val Asp Leu Pro 35 4045 Tyr Pro Leu Asn Asn Phe Ser Val Ala Lys Cys Gln Leu Met Lys Thr 50 5560 Glu Arg Pro Lys Pro Asn Thr Phe Ile Ile Arg Cys Leu Gln Trp Thr 65 7075 80 Thr Val Ile Glu Arg Thr Phe His Val Asp Thr Pro Glu Glu Arg Glu 8590 95 Glu Trp Thr Glu Ala Ile Gln Ala Val Ala Asp Arg Leu Gln Arg Gln100 105 110 Glu Glu Glu Arg Met Asn Cys Ser Pro Thr Ser Gln Ile Asp AsnIle 115 120 125 Gly Glu Glu Glu Met Asp Ala Ser Thr Thr His His Lys ArgLys Thr 130 135 140 Met Asn Asp Phe Asp Tyr Leu Lys Leu Leu Gly Lys GlyThr Phe Gly 145 150 155 160 Lys Val Ile Leu Val Arg Glu Lys Ala Ser GlyLys Tyr Tyr Ala Met 165 170 175 Lys Ile Leu Lys Lys Glu Val Ile Ile AlaLys Asp Glu Val Ala His 180 185 190 Thr Leu Thr Glu Ser Arg Val Leu LysAsn Thr Arg His Pro Phe Leu 195 200 205 Thr Ser Leu Lys Tyr Ser Phe GlnThr Lys Asp Arg Leu Cys Phe Val 210 215 220 Met Glu Tyr Val Asn Gly GlyGlu Leu Phe Phe His Leu Ser Arg Glu 225 230 235 240 Arg Val Phe Ser GluAsp Arg Thr Arg Phe Tyr Gly Ala Glu Ile Val 245 250 255 Ser Ala Leu AspTyr Leu His Ser Gly Lys Ile Val Tyr Arg Asp Leu 260 265 270 Lys Leu GluAsn Leu Met Leu Asp Lys Asp Gly His Ile Lys Ile Thr 275 280 285 Asp PheGly Leu Cys Lys Glu Gly Ile Thr Asp Ala Ala Thr Met Lys 290 295 300 ThrPhe Cys Gly Thr Pro Glu Tyr Leu Ala Pro Glu Val Leu Glu Asp 305 310 315320 Asn Asp Tyr Gly Arg Ala Val Asp Trp Trp Gly Leu Gly Val Val Met 325330 335 Tyr Glu Met Met Cys Gly Arg Leu Pro Phe Tyr Asn Gln Asp His Glu340 345 350 Lys Leu Phe Glu Leu Ile Leu Met Glu Asp Ile Lys Phe Pro ArgThr 355 360 365 Leu Ser Ser Asp Ala Lys Ser Leu Leu Ser Gly Leu Leu IleLys Asp 370 375 380 Pro Asn Lys Arg Leu Gly Gly Gly Pro Asp Asp Ala LysGlu Ile Met 385 390 395 400 Arg His Ser Phe Phe Ser Gly Val Asn Trp GlnAsp Val Tyr Asp Lys 405 410 415 Lys Leu Val Pro Pro Phe Lys Pro Gln ValThr Ser Glu Thr Asp Thr 420 425 430 Arg Tyr Phe Asp Glu Glu Phe Thr AlaGln Thr Ile Thr Ile Thr Pro 435 440 445 Pro Glu Lys Cys Gln Gln Ser AspCys Gly Met Leu Gly Asn Trp Lys 450 455 460 Lys 465

We claim:
 1. A mutant Chrysosporium strain comprising a nucleic acidsequence encoding a polypeptide of interest, said nucleic acid sequencebeing operably linked to an expression-regulating region and optionallya secretion signal sequence, said mutant strain expressing saidpolypeptide of interest at a higher level than the correspondingnon-mutant strain under the same conditions.
 2. A mutant Chrysosporiumstrain according to claim 1, said mutant being obtained by recombinantmethods comprising stable introduction of at least one heterologousnucleic acid sequence selected from heterologous polypeptide-encodingnucleic acid sequences, heterologous signal sequences and heterologousexpression-regulating sequences.
 3. A mutant Chrysosporium strainaccording to claim 2, wherein said polypeptide of interest is aheterologous polypeptide of plant, animal (including human), insect,algal, bacterial, archaebacterial or fungal origin.
 4. A mutantChrysosporium strain according to claim 1, wherein said polypeptide ofinterest is a homologous polypeptide which is expressed at a higherlevel than in the corresponding non-mutant strain under the sameconditions.
 5. A mutant Chrysosporium strain according to claim 1,wherein said polypeptide of interest is selected fromcarbohydrate-degrading enzymes, proteases, lipases, esterases, otherhydrolases, oxidoreductases and transferases.
 6. A mutant Chrysosporiumstrain according to claim 1, wherein said polypeptide of interest isselected from fugal enzymes allowing (over)production of primarymetabolites, including organic acids, and secondary metabolites,including antibiotics.
 7. A mutant Chrysosporium strain according toclaim 1, wherein said polypeptide of interest is inactivated at a pHbelow
 6. 8. A mutant Chrysosporium strain according to claim 1, whereinsaid polypeptide of interest exhibits optimal activity and/or stabilityat a pH above 6, and/or has more than 70% of its activity and/orstability at a pH above
 6. 9. A mutant Chrysosporium strain according toclaim 1, comprising a heterologous signal sequence.
 10. A mutantChrysosporium strain according to claim 1, comprising a fungal signalsequence.
 11. A mutant Chrysosporium strain according to claim 10,wherein the fungal signal sequence is a signal sequence of a cellulase,β-galactosidase, xylanase, pectinase, esterase, protease, amylase,polygalacturonase or hydrophobin.
 12. A mutant Chrysosporium strainaccording to claim 1, further comprising a selectable marker.
 13. Amutant Chrysosporium strain according to claim 12, wherein theselectable marker confers resistance to a drug or relieves a nutritionaldefect.
 14. A mutant Chrysosporium strain according to claim 1,comprising a heterologous expression-regulating region.
 15. A mutantChrysosporium strain according to claim 1, comprising a fungalexpression-regulating region.
 16. A mutant Chrysosporium strainaccording to claim 15, wherein the expression-regulating regioncomprises is an inducible promoter.
 17. A mutant Chrysosporium strainaccording to claim 15, wherein the expression-regulating regioncomprises a high expression promoter.
 18. A mutant Chrysosporium strainaccording to claim 1, said mutant being obtained by mutagenesis steps,the steps including at least one step chosen from the group consistingof UV irradiation and chemical mutagenesis.
 19. A mutant Chrysosporiumstrain according to claim 18, wherein the mutagenesis steps comprise afirst UV irradiation step, a N-methyl-N′-nitro-N-nitrosoguanidinetreatment step, and a second UV irradiation step.
 20. A mutantChrysosporium strain according to claim 1, said mutant being derivedfrom Chrysosporium lucknowense.
 21. A mutant Chrysosporium strainaccording to claim 20, wherein said mutant is or is derived from aChrysosporium lucknowense mutant strain selected from the groupconsisting of C. lucknowense strain C1 (VKM F-3500 D), UV13-6 (VKMF-3632 D), NG7C-19 (VKM F-3633 D), and UV18-25 (VKM F-3631 D).
 22. Amutant Chrysosporium strain according to claim 1, wherein, when aTrichodenna reesei strain and said Chrysosporium strain are culturedunder equivalent optimal conditions, when the Trichoderma cultureattains a viscosity of 200-600 cP, said Chrysosporium strain exhibits abiomass of less than half that of the Trichoderma.
 23. A mutantChrysosporium strain according to claim 1, said strain producing atleast the amount of cellulase in moles per liter as produced by any ofthe Chrysosporium lucknowense mutant strains C1 (VKM F-3500 D), UV13-6(VKM F-3632 D), NG7C-19 (VKM F-3633 D), and UV18-25 (VKM F-3631 D). 24.A mutant Chrysosporium strain according to claim 1, said strainproducing less protease than produced by the Chrysosporium lucknowensestrain C1 (VKM F-3500 D).
 25. A mutant Chrysosporium strain according toclaim 24, wherein the strain produces less than half the amount ofprotease produced by the C1 strain.
 26. A nucleic acid constructcomprising a nucleic acid expression-regulatory region derived fromChrysosporium lucknowense, operably linked to a polypeptide-encodingnucleic acid sequence.
 27. A nucleic acid construct acording to claim26, wherein the expression-regulatory region is derived from aChrysosporium lucknowense strain selected from the group consisting ofC1 (VKM F-3500 D) and UV18-25 (VKM F-3631 D).
 28. A nucleic acidconstruct according to claim 26, said expression-regulatory regioncomprising a promoter sequence associated with cellulase expression,xylanase expression, or gpdA expression.
 29. A recombinant microorganismcontaining a nucleic acid construct according to claim 26, and capableof expressing the polypeptide encoded by the coding nucleic acidsequence.
 30. The microorganism of claim 29, wherein the microorganismis a fungal strain.
 31. A method of producing a polypeptide of interest,comprising culturing a strain according to claim 1 under conditionspermitting expression of the protein or polypeptide, and recovering thesubsequently produced polypeptide of interest.
 32. The method of claim31, wherein the conditions further permit secretion of the protein orpolypeptide of interest.
 33. A method of producing a polypeptide ofinterest, said method comprising culturing a strain according to claim29 under conditions permitting expression of the protein or polypeptideand recovering the subsequently produced polypeptide of interest. 34.The method of claim 33, wherein the conditions further permit secretionof the protein or polypeptide of interest.
 35. A method according to anyone of claims 31-34, wherein the protein or polypeptide is expressed asa precursor protein, further comprising the step of cleavage of theprecursor into the polypeptide or precursor of interest.
 36. The methodaccording to claim 35, wherein the cleavage step is cleavage with anenzyme selected from the group consisting of Kex-2 like proteases, basicamino acid paired proteases, or Kex-2.
 37. The method according to claim33, wherein the cultivation occurs at pH in the range 6-9, and/or at atemperature between 25 and 43° C.
 38. A method for producing a mutantChrysosporium strain according to claim I comprising stably introducinga nucleic acid sequence encoding a heterologous or homologouspolypeptide into a Chrysosporium strain, said nucleic acid sequencebeing operably linked to an expression regulating region.
 39. A methodaccording to claim 38, wherein the nucleic acid is introduced by theprotoplast transformation method.
 40. A protein corresponding to aChrysosporium glycosyl hydrolase family 7, exhibiting at least 75% aminoacid identity as determined by the BLAST algorithm with the amino acidsequence of SEQ ID NO: 1 or a part thereof having at least 20 contiguousamino acids which are identical to the corresponding part of the aminoacid sequence 1-246 or 394-526 of SEQ ID NO:
 1. 41. A proteincorresponding to a Chrysosporium glycosyl hydrolase family 10,exhibiting at least 70% amino acid identity as determined by the BLASTalgorithm with the amino acid sequence of SEQ ID NO:2 or a part thereofhaving at least 20 contiguous amino acids which are identical to thecorresponding part of the amino acid sequence 1-383 of SEQ ID NO:2. 42.A protein corresponding to a Chrysosporium glycosyl hydrolase family 10,exhibiting at least 65% amino acid identity as determined by the BLASTalgorithm with the amino acid sequence of SEQ ID NO:2 or a part thereofhaving at least 20 contiguous amino acids which are identical to thecorresponding part of the amino acid sequence 1-383 of SEQ ID NO:2, 43.A protein corresponding to a Chrysosporium glycosyl hydrolase family 10and comprising a cellulose-binding domain selected from the groupcomprising (a) domains having at least 75% amino acid identity with theamino acid sequence 22-53, and (b) domains having at least 20 contiguousamino acids identical to a part of amino acid sequence 22-53 of SEQ IDNO:2.
 44. A fungal glycosyl hydrolase of family 10, comprising acellulose-binding domain, not derived from Fusarium oxysporum.
 45. Aprotein corresponding to a Chrysosporium glycosyl hydrolase family 12,exhibiting at least 65% amino acid identity as determined by the BLASTalgorithm with the amino acid sequence of SEQ ID NO:3 or a part thereofhaving at least 20 contiguous amino acids which are identical to thecorresponding part of the amino acid sequence 1-247 of SEQ ID NO:3. 46.A protein corresponding to a Chrysosporium glyceraldehyde phosphatedehydrogenase, exhibiting at least 86% amino acid identity as determinedby the BLAST algorithm with the partial amino acid sequence of SEQ IDNO:4 or a part thereof having at least 20 contiguous amino acids whichare identical to the corresponding part of the amino acid sequence 1-277of SEQ ID NO:4.
 47. A protein corresponding to a Chrysosporium glycosylhydrolase family 45, exhibiting at least 83% amino acid identity asdetermined by the BLAST algorithm with the amino acid sequence of SEQ IDNO:5 or a part thereof having at least 20 contiguous amino acids whichare identical to the corresponding part of the amino acid sequence 1-225of SEQ ID NO:5.
 48. A protein corresponding to a Chrysosporium glycosylhydrolase family 6, exhibiting at least 50% amino acid identity asdetermined by the BLAST algorithm with the amino acid sequence of SEQ IDNO:6 or a part thereof having at least 20 contiguous amino acids whichare identical to the corresponding part of the amino acid sequences1-395 of SEQ ID NO:6.
 49. A method for hydrolysing β-glucosidic bonds,comprising contacting a β-glucoside with an enzyme according to any oneof claims 40, 45, 47, and
 48. 50. A method for hydrolysing β-xylosidicbonds, comprising contacting a β-xyloside with an enzyme according toclaim
 41. 51. A nucleic acid sequence encoding a protein according toclaim
 40. 52. A nucleic acid sequence encoding a protein according toclaim
 41. 53. A nucleic acid sequence encoding a protein according toclaim
 42. 54. A nucleic acid sequence encoding a protein according toclaim
 45. 55. A nucleic acid sequence encoding a protein according toclaim
 46. 56. A nucleic acid sequence encoding a protein according toclaim
 47. 57. A nucleic acid sequence encoding a protein according toclaim
 48. 58. A nucleic acid sequence comprising at least 70% of thenucleotides contained in the 5′-noncoding region of the nucleic acidsequence of any one of SEQ ID NOS: 1-6.
 59. A nucleic acid constructcomprising a nucleic acid expression-regulatory region derived fromChrysosporium, contained in the 5′-noncoding region of the nucleic acidsequence of any one of SEQ ID No's 1-6, operationally linked to anucleic acid sequence encoding a polypetide of interest.
 60. Arecombinant microbial strain, containing a nucleic acid constructaccording to claim 59, and capable of expressing the polypeptide encodedby the coding nucleic acid sequence.
 61. A recombinant microbial strainaccording to claim 60, which is a fungal strain.
 62. A recombinantmicrobial strain, containing a nucleic acid sequence according to claim51, and capable of expressing the polypeptide encoded by the codingnucleic acid sequence.
 63. A recombinant microbial strain, containing anucleic acid sequence according to claim 52 and capable of expressingthe polypeptide encoded by the coding nucleic acid sequence.
 64. Arecombinant microbial strain, containing a nucleic acid sequenceaccording to claim 53, and capable of expressing the polypeptide encodedby the coding nucleic acid sequence.
 65. A recombinant microbial strain,containing a nucleic acid sequence according to claim 54, and capable ofexpressing the polypeptide encoded by the coding nucleic acid sequence.66. A recombinant microbial strain, containing a nucleic acid sequenceaccording to claim 55, and capable of expressing the polypeptide encodedby the coding nucleic acid sequence.
 67. A recombinant microbial strain,containing a nucleic acid sequence according to claim 56, and capable ofexpressing the polypeptide encoded by the coding nucleic acid sequence.68. A recombinant microbial strain, containing a nucleic acid sequenceaccording to claim 57, and capable of expressing the polypeptide encodedby the coding nucleic acid sequence.
 69. A process of producing apolypeptide using a construct according to claim
 59. 70. A process ofproducing a polypeptide using a microbial strain according to any one ofclaims 60-68.
 71. An oligonucleotide probe comprising at least 15contiguous nucleotides of the nucleic acid sequence of any one of SEQ IDNo's 1-6, or the complement thereof.
 72. An oligonucleotide probeaccording to claim 71, wherein the probe is 20-50 nucleotides in length.73. An oligonucleotide probe according to either one of claims 71 or 72,wherein the probe is labelled with a detectable label.